The Effects of Acute and Chronic Beetroot Juice Supplementation on 1 Exercise Economy and Time Trial Performance in Recreationally 2

Active Females 3 4

by 5 6

Kate Aiko Wickham 7 8 9 10 11 12 13 14

A Thesis 15 Presented to 16

The University of Guelph 17 18 19 20 21 22 23

In partial fulfillment of requirements 24 for the degree of 25

Masters of Science 26 in 27

Human Health and Nutritional Sciences 28 29 30 31 32 33 34 35 36

Guelph, Ontario, Canada 37 38

© Kate Aiko Wickham, July 2018 39

i

ABSTRACT 40 41 42

THE EFFECTS OF ACUTE AND CHRONIC BEETROOT JUICE 43 SUPPLEMENTATION ON EXERCISE ECONOMY AND TIME TRIAL 44

PERFORMANCE IN RECREATIONALLY ACTIVE FEMALES 45 46 47

Kate Aiko Wickham Advisor: 48 University of Guelph, 2018 Dr. Lawrence L. Spriet 49

50

Beetroot juice (BRJ) is a supplement that reduces the oxygen cost of submaximal 51

exercise and improves performance in recreationally active males, both acutely and chronically. 52

However, evidence supporting the effects of BRJ in females is lacking. This thesis examined the 53

effects of acute and chronic BRJ supplementation in 12 recreationally active females (VO2peak: 54

40.6 + 1.2 mL O2/kg/min). Using a randomized, double-blind, placebo-controlled, crossover 55

design, subjects supplemented with 280 mL BRJ or a nitrate-free placebo for 8 days separated by 56

a 9 + 1 days washout period. On days 1 (acute) and 8 (chronic), subjects completed a 57

submaximal cycling protocol and a 4 kJ/kg time trial. Acute and chronic BRJ supplementation 58

had no effect on VO2 at 50 or 70% VO2peak, despite large increases in plasma nitrate and nitrite. 59

Additionally, there was no difference in time trial completion. To conclude, recreationally active 60

females may not benefit from acute or chronic BRJ supplementation.61

iii

Acknowledgements 62

63

Thank you to my parents for their unwavering support throughout my academic and life 64

journey. Dad, thank you for teaching me to persevere and to work diligently in the pursuit of my 65

dreams. Mom, thank you for teaching me to carry myself with poise and a kind heart. I owe my 66

character and my successes to you both. 67

68

Thank you to my friends, near and far, for encouraging me to lead my best life and for 69

being by my side throughout my personal growth as an academic and an individual. Your life 70

stories, and the stories we share together, have had a profound impact on my life and have helped 71

shape my values and passions. 72

73

Thank you to my MSc advisor, Dr. Lawrence Spriet, for being a steadfast role model. 74

You have taught me how powerful the combination of charisma, intelligence, tenacity, and 75

compassion can be. Thank you for the countless opportunities and challenges you have provided 76

me with. These moments have reinforced my passions and have encouraged me to continue my 77

mission to pursue a career merging the worlds of sport, academia and industry. 78

79

Thank you to my current and past Spriet Lab family including Devin McCarthy, Jamie 80

Pereira, Sebastian Jannas, Jamie Whitfield, Tyler Vermeulen, Alex Gamble, and Jessica Bigg. I 81

am thankful that I had the privilege to share the last two years of adventure with you. Thank you 82

to my HHNS family for welcoming me with open arms and providing me with an unforgettable 83

graduate school experience. 84

85

Lastly, thank you to my research participants for their dedication to my studies. Your 86

willingness to drink beetroot juice for weeks, to abstain from caffeine on several occasions, and 87

to push yourselves to your physical limits – all completed with smiles on your faces – did not go 88

unnoticed or unappreciated. 89

iv

Table of Contents 90

Abstract ……………………………………………………………………………… i 91

Acknowledgements ……………………………………………………………… ii 92

Table of Contents ………………………………………………………………… iii - v 93

List of Tables ……………………………………………………………………… vi 94

List of Figures …………………………………………………………………… vii - viii 95

List of Symbols and Abbreviations ………………………………………………. ix - x 96

Chapter 1: Review of Literature ………………………………………………… 1 - 39 97 98

Nitric Oxide: Roles, Origins, and Targets …………………………………………… 1 - 4 99

Beetroot Juice Pharmacokinetics, Dose, Duration …………………………………. 4 - 10 100

Dietary Nitrate Pharmacokinetics ………………………………………………………… 4 - 5 101 Dose Response: Plasma Levels, Blood Pressure, Oxygen Uptake, and Performance …… 5 - 7 102 Acute vs. Chronic Supplementation: Plasma Levels, Oxygen Uptake and Performance … 8 - 10 103

104 The Effects of Beetroot Juice Supplementation on Exercise Economy and Athletic 105 Performance ……………………………………………………………………… 10 - 20 106

107 Moderate Intensity Submaximal Exercise Economy ……………………………. 10 - 12 108

Severe and Maximal Intensity Exercise Economy ………………………………. 12 - 13 109

Time Trial Performance ………………………………………………………… 14 - 16 110

Aerobic Cycling Time Trial Performance ……………………………………. 14 - 15 111 Aerobic Running Time Trial Performance …………………………………… 15 - 16 112 Water Sport Time Trial Performance ……………………………………………… 16 113

Time to Exhaustion …………………………………………………………………………. 17 114 Sprint and Team Sport Performance …………………………………………………. 17 - 18 115 Strength Performance ………………………………………………………………………. 18 116 Performance During Hypoxia ……………………………………………………………. 19 117 Conclusion …………………………………………………………………………………. 20 118

119 Individual Responses to Beetroot Juice Supplementation ……………………… 20 - 24 120

Fitness Status …………………………………………………………………………. 21 - 23 121 Diet ………………………………………………………………………………………… 23 122 Digestive Pathway …………………………………………………………………… 23 - 24 123 124

v

Beetroot Juice Improves Exercise Economy and Performance - Potential Mechanisms of 125 Action …………………………………………………………………………… 24 - 32 126

Are We Sure It's Nitrate? ……………………………………………………………… 24 - 26 127 Improved Blood Flow ………………………………………………………………… 26 128 Improved Mitochondrial Function …………………………………………………… 27 - 29 129

Reduced Oxygen Cost of Mitochondrial ATP Resynthesis ………………… 27 - 28 130 Mitochondrial Biogenesis …………………………………………… 28 - 29 131

Improved Contractile Efficiency ………………………………………………… 29 - 32 132 Reduced ATP Cost of Skeletal Muscle Force Production …………………… 29 - 31 133 Reactive Oxygen Species ……………………………………………………… 31 - 32 134 135

Women - Addressing the Gender Gap …………………………………………… 32 - 39 136 Characterization of the Menstrual Cycle ……………………………………………………. 33 137 Nitric Oxide Varies Throughout the Menstrual Cycle ………………………………… 34 - 35 138 Pharmacokinetics of Hormonal Contraceptives …………………………………………… 35 139 Previous Female Beetroot Juice Literature …………………………………………… 36 - 39 140 Conclusions ………………………………………………………………………………. 39 141

Chapter 2: Statement of the Problem …………………………………………… 40 - 42 142

Rationale ………………………………………………………………………… 40 - 41 143

Hypotheses ………………………………………………………………………. 41 - 42 144

Chapter 3: Methods ……………………………………………………………… 42 - 50 145

Subjects ………………………………………………………………………………. 42 146

Study Design ……………………………………………………………………. 42 - 43 147

Pre-Experimental Tests ………………………………………………………… 43 - 44 148

Diet, Sleep and Exercise Standardization ……………………………………………. 45 149

Experimental Exercise Protocol ……………………………………………………… 46 150

Blood Sampling and Analysis ……………………………………………………. 47 - 49 151

Compliance …………………………………………………………………………… 49 152

Statistical Analysis ………………………………………………………………. 49 - 50 153

Chapter 4: Results ………………………………………………………………… 50 - 61 154

Blood Parameters ………………………………………………………………… 50 - 52 155

Pre-Trial Characteristics ………………………………………………………………. 52 156

Environmental Characteristics ………………………………………………………… 53 157

Hydration Status ………………………………………………………………………. 53 158

Exercise Economy 50% VO2peak ………………………………………………… 53 - 54 159

vi

Exercise Economy 70% VO2peak ………………………………………………… 55 - 56 160

Time Trial Performance ……………………………………………………………… 57 161

Compliance, Blinding, and Adverse Events ……………………………………… 60 - 61 162

Chapter 5: Discussion …………………………………………………………… 63 - 75 163

Comparison to Previous Work in Women ………………………………………. 63 - 65 164

Nitrate Digestion …………………………………………………………………. 65 - 67 165

Nitrate and Nitrite Levels ……………………………………………………………. 67 166

Antioxidant Capacity ………………………………………………………………… 68 167

Differences in Skeletal Muscle Fiber Types and Capillarization ………………… 69 - 70 168

Mitochondrial Efficiency …………………………………………………………. 70 - 71 169

Calcium-Handling Proteins ………………………………………………………. 71 - 72 170

Individual Responders ……………………………………………………………. 72 - 73 171

Limitations ……………………………………………………………………………… 73 172

Future Directions …………………………………………………………………. 74 - 75 173

Conclusion ……………………………………………………………………………. 75 174

References …………………………………………………………………………… 76 - 87 175 176

vi

List of Tables 177

Table 1 - Respiratory variables at 50% and 70% VO2peak following acute and chronic 178 supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values reported as 179 mean + SEM. *, Chronic trials demonstrated significantly higher carbon dioxide production 180 (VCO2) compared to acute trials. **, BRJ significantly decreased ventilation (VE) compared to 181 PLA. 61 182 183 Table 2 - Heart rate (HR), rating of perceived exertion (RPE) and revolutions per minute (rpm) 184 recorded at 5 and 10 min at 50% and 70% peak oxygen consumption (VO2peak) following acute 185 and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values 186 reported as mean + SEM. 62 187

188

vii

List of Figures 189

Figure 1 - An illustration of the oxygen-independent and oxygen-dependent pathways for nitric 190 oxide production. 2 191 192 Figure 2 - The pharmacokinetic profile and dose response relationship of 4.2, 8.4 and 16.8 mM 193 dietary nitrate on plasma NO3- and nitrite. 194 Error! Bookmark not defined. 195 196 Figure 3 - An illustration of the effects of beetroot juice supplementation on oxygen uptake 197 kinetics from rest to moderate intensity exercise in recreationally active males. 12 198 199 Figure 4 - An illustration of the effects of beetroot juice supplementation on oxygen uptake 200 kinetics during moderate intensity exercise in simulated altitude (15% oxygen) in recreationally 201 active males. 202 Error! Bookmark not defined. 203 204 Figure 5 - Hormonal profile throughout a normal menstrual cycle with significant surges in 205 estrogen and progesterone. 206 Error! Bookmark not defined. 207 208 Figure 6 - Low estrogen and progesterone levels throughout the menstrual cycle in women 209 actively using two different forms of hormonal birth control. 35 210 211 Figure 7 - A schematic of the experimental design consisting of 7 laboratory visits. 43 212 213 Figure 8 - Experimental trial exercise protocol. 47 214

215 Figure 9 - Plasma nitrate at baseline (B) and two hours post-ingestion (2 h) of 140 mL beetroot 216 juice or nitrate-free placebo both acutely and chronically. 217 Error! Bookmark not defined. 218 219 Figure 10 - Plasma nitrite at baseline (B) and two hours post-ingestion (2 h) of 140 mL beetroot 220 juice or nitrate-free placebo both acutely and chronically. 221 Error! Bookmark not defined. 222 223 Figure 11 - Mean oxygen uptake at 50% peak oxygen uptake following acute and chronic 224 supplementation with beetroot juice or a nitrate-free placebo. 55 225 226 Figure 12 - Individual oxygen uptake at 50% peak oxygen uptake following acute and chronic 227 supplementation with beetroot juice or a nitrate-free placebo. 55 228 229 Figure 13 - Mean oxygen uptake at 70% peak oxygen uptake following acute and chronic 230 supplementation with beetroot juice or a nitrate-free placebo. 56 231 232

viii

Figure 14 - Individual oxygen uptake at 70% peak oxygen uptake following acute and chronic 233 supplementation with beetroot juice or a nitrate-free placebo. 57 234 235 Figure 15 - Time trial learning effect. 58 236 237 Figure 16 - Mean time trial completion. 58 238 Figure 17 - Mean time to complete each 20% split of the time trial. 59 239 240 Figure 18 - Mean heart rate at the end of each 20% split during the time trial. 59 241 242 Figure 19 - Mean rating of perceived exertion at the end of each 20% split during the time trial. 243

60 244 245 Figure 20 - Mean power output at the end of each 20% split during the time trial. 60 246

247

ix

List of Symbols and Abbreviations 248

ADP - Adenosine Diphosphate 249

ANT - Adenine Nucleotide Translocator 250

ATP - Adenosine Triphosphate 251

BH4 - Tetrahydrobiopterin 252

BRJ - Beetroot Juice 253

Ca2+ - Calcium 254

cGMP - Cyclic Guanosine Monophosphate 255

COXIV - Cytochrome Oxidase IV 256

CV - Coefficient of Variance 257

DBP - Diastolic Blood Pressure 258

ETC - Electron Transport Chain 259

FAD - Flavin Adenine Dinucleotide 260

FMN - Flavin Mononucleotide 261

Gc - Guanylyl Cyclase 262

H+ - Hydrogen Ion 263

HR - Heart Rate 264

KNO3 - Potassium Nitrate 265

MAP - Mean Arterial Pressure 266

NADPH - Nicotinamide-Adenine-Dinucleotide-Phosphate 267

NO3- - Nitrate 268

NaNO3 - Sodium Nitrate 269

NO - Nitric Oxide 270

x

NOS - Nitric Oxide Synthase 271

NO2- - Nitrite 272

O2 - Oxygen 273

PCr - Phosphocreatine 274

PGC1a - Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-Alpha 275

PLA - Placebo 276

PO - Power Output 277

P/O Ratio - Phosphate/Oxygen Ratio (ATP synthesis/Oxygen utilization) 278

ROS - Reactive Oxygen Species 279

RPE - Rating of Perceived Exertion 280

SBP - Systolic Blood Pressure 281

SERCA - Sarcoplasmic Reticulum Calcium ATPase 282

SOD - Superoxide Dismutase 283

TFAM - Mitochondrial Transcription Factor A 284

TT - Time Trial 285

UCP-3 - Uncoupling Protein 3 286

UVA - Ultraviolet A 287

VO2 - Oxygen Uptake 288

VO2max - Maximal Oxygen Uptake 289

VO2peak - Peak Oxygen Uptake 290

W - Watts291

1

Chapter 1: Review of the Literature 292

Nitric Oxide: Roles, Origins and Targets 293

Nitric oxide (NO) is a gaseous signalling molecule that elicits potent effects on numerous 294

biological tissues and is involved in an array of physiological processes including, but not 295

limited to: vasodilation, calcium handling, mitochondrial respiration, and neurotransmission 296

(Bailey et al. 2011). 297

NO can be produced endogenously and obtained from exogenous dietary sources. 298

Endogenously, NO is produced through the oxidation of L-arginine to L-citrulline and NO 299

(Figure 1). This reaction requires oxygen (O2) and reduced nicotinamide-adenine-dinucleotide 300

phosphate (NADPH) as a co-substrate as well as flavin adenine dinucleotide (FAD), flavin 301

mononucleotide (FMN), tetrahydrobiopterin (BH4), heme, and calmodulin as cofactors and 302

substrates and is catalyzed by the enzyme nitric oxide synthase (NOS) (Bailey et al. 2011). NOS 303

exists in several isoforms depending on its location and role within the biological system of 304

interest. These isoforms include neuronal NOS, endothelial NOS, and inducible NOS. These 305

isoforms will not be discussed in detail as they are beyond the scope of this literature review. 306

In 1994, it was acknowledged by Benjamin et al. (1994) as well as Lundberg et al. (1994) 307

that NO can be produced through an alternative exogenous pathway that does not rely on O2. 308

This anaerobic pathway involves the sequential one electron reduction of nitrate (NO3-) to nitrite 309

(NO2-) and further to NO (Figure 1). This allows the formation of NO in situations where the 310

NOS enzyme may be impaired such as hypoxia or disease. This pathway has important 311

implications for cardiovascular disease as well as exercise performance. The NO3- necessary for 312

this reaction is obtained primarily from dietary sources such as green leafy vegetables (spinach, 313

2

lettuce, kale, arugula) and root vegetables (beetroot, yams, turnips, carrots). Spinach, lettuce and 314

beetroot have the highest dietary NO3- concentrations (Hord et al. 2009). 315

Approximately 75% of ingested dietary NO3- is excreted in the urine, with only ~25% 316

entering the enterosalivary circulation (Webb et al. 2008). Dietary NO3- is swallowed and 317

subsequently absorbed from the stomach and small intestines. Once it enters the circulation, it is 318

concentrated in the salivary glands. NO3- is secreted from the salivary glands into the mouth via 319

saliva, and anaerobic bacteria facilitate the reduction of NO3- to NO2-. The NO2- is swallowed 320

again and is further reduced to NO in the acidic environment of the stomach (Bailey et al. 2011). 321

322

Figure 1: The oxygen (O2)-independent (left) and O2-dependent (right) pathways for nitric oxide 323 (NO) production. The O2-independent pathway relies on dietary nitrate (NO3-) to undergo 324 sequential reduction to nitrite (NO2-) and to NO. The O2-dependent pathway relies on a number 325 of substrates and cofactors including nicotinamide adenine dinucleotide phosphate (NADPH), 326 flavin adenine nucleotide (FAD), tetrahydrobiopterin (BH4), haem, and calmodulin as well as the 327 enzyme nitric oxide synthase (NOS). This figure also depicts an array of the putative biological 328 targets of NO (Bailey et al. 2011). 329 330

A final and newly regarded pathway by which the body generates NO is through ultra 331

violet A (UVA) exposure (Oplander et al. 2009; Liu et al. 2014; Muggeridge et al. 2015). The 332

NOS enzyme is active in a number of skin cells such as keratinocytes, melanocytes, fibroblasts 333

3

and endothelial cells (Bruch-Gerharz et al. 1998). The NO produced by skin cell NOS are 334

converted to nitroso compounds, NO2- or NO3- (Oplander et al. 2009). Remarkably, when UVA 335

rays penetrate the skin they can convert these compounds back to NO. This may help explain 336

why blood pressure is shown to be lower in countries that are closer to the equator and may 337

partially explain why blood pressure is typically lower in the summer months compared to the 338

winter (Oplander et al. 2009). 339

A plethora of nutritional supplements have been used in an attempt to augment the human 340

body’s production of NO. L-arginine is able to accumulate in the blood, but there is equivocal 341

evidence as to whether it increases NO production in healthy individuals (Bode-Böger et al. 342

1998; Liu et al. 2009; Bailey et al. 2010; Alvares et al. 2012). Furthermore, it has been shown 343

that citrulline, the co-product of NO synthesis, can be enzymatically converted back to arginine 344

to produce NO (Schwedhelm et al. 2007; Bryk et al. 2008). These supplements provide substrate 345

for the endogenous production of NO via the oxidation of L-arginine, however a greater body of 346

literature is required to support the effectiveness of these supplements. Concentrated beetroot 347

juice (BRJ) is a potent source of dietary NO3- containing 6.5 mmol (mM) or 400 mg per 70 mL 348

dose. BRJ facilitates the production of NO through the exogenous O2-independent NO3--NO2--349

NO pathway and has been shown to elevate plasma NO2- in a number of studies (Vanhatalo et al. 350

2010; Wylie et al. 2013; Bailey et al. 2015; Betteridge et al. 2016). Similarly, sodium nitrate 351

(NaNO3) and potassium nitrate (KNO3) administered as supplemental forms of NO3- can 352

augment the NO3--NO2--NO pathway. NaNO3 has been shown to elevate plasma NO2- in a 353

number of studies (Larsen et al. 2010; Bescós et al. 2012; Gasier et al. 2017), and KNO3 has also 354

been shown to increase plasma NO2- following supplementation (Kapil et al. 2010). Therefore, 355

the literature supports the use of dietary NO3- to increase plasma [NO3-] and [NO2-]. 356

4

NO is an unstable molecule and has a very short half-life in the blood of merely a few 357

milliseconds (Kelm 1999). NO is rapidly oxidized to NO2-, and this is the largest circulating pool 358

of NO3- products in the blood (Cosby et al. 2003). NO2- can be converted back to NO for cellular 359

signalling or can be further oxidized to NO3-. NO can act on several cellular targets including 360

proteins that contain transition metals or thiol groups (Tengan et al. 2013). These mechanisms 361

will be addressed in detail in a subsequent section of this literature review. 362

BRJ Pharmacokinetics, Dose and Duration 363

Dietary NO3- Pharmacokinetics 364

Pharmacokinetic profiles are essential to nutritional intervention studies as they provide a 365

framework to validate the accumulation of the treatment in the blood, demonstrate the time 366

course with which the treatment rises and peaks in the blood, and depict the rate of clearance 367

from the body. Previous research has demonstrated that plasma NO3- levels begin to rise ~30 min 368

post-ingestion and peak ~1.5 h following the ingestion of BRJ (Webb et al. 2008; Wylie et al. 369

2013) or KNO3 (Kapil et al. 2010). Plasma NO3- remains elevated for up to 24 h following a 370

single dose of dietary NO3- (Wylie et al. 2013; Kapil et al. 2010). Furthermore, plasma NO2- 371

levels begin to rise ~1.5 h post-ingestion, peaking ~2.5 h post-ingestion, and have been shown to 372

remain elevated for an additional 3 h (Webb et al. 2008; Kapil et al. 2010; Wylie et al. 2013). 373

Both plasma NO3- and NO2- have been shown to return to baseline concentrations ~24 h 374

following a large, acute dose of dietary NO3- (Webb et al. 2008; Kapil et al. 2010; Wylie et al. 375

2013). However, plasma NO2- levels have been shown to decrease by 12 h post-ingestion with a 376

moderate dose (8.4 mM) of dietary NO3- (Wylie et al. 2013). Interestingly, the slight lag time for 377

peak plasma NO2- compared to plasma NO3- likely reflects the time required for NO3- to enter the 378

enterosalivary circulation and undergo the sequential reduction from NO3- to NO2-. The results 379

5

from this study indicate that dietary NO3- should be administered every 12 h to ensure plasma 380

[NO3-] and [NO2-] remain chronically elevated above baseline concentrations. 381

The conversion in the enterosalivary circulation can be disrupted by spitting (Webb et al. 382

2008) or destruction of the anaerobic bacteria via antibacterial mouthwash (Govoni et al. 2008; 383

McDonagh et al. 2015; Woessner et al. 2016). As expected, when the enterosalivary circulation 384

is disrupted following a dose of dietary NO3- there is a concomitant termination in the reduction 385

of NO3- to NO2-, as represented by no change in plasma [NO2-] despite a significant increase in 386

plasma [NO3-]. Furthermore, since plasma [NO2-] is correlated with NO production, the 387

disruption of the enterosalivary circulation eliminates the beneficial biological effects associated 388

with dietary NO3- supplementation (Webb et al. 2008). This demonstrates that oral bacteria are 389

fundamental to the rise in plasma [NO2-] and subsequent biological effects following a dose of 390

dietary NO3-. 391

Dose Response: Plasma Levels, Blood Pressure, Oxygen Uptake (VO2) and Performance 392

To date, only two studies have investigated the dose-response relationship associated 393

with dietary NO3- supplementation. Kapil et al. (2010) investigated the dose-response associated 394

with supplementing acutely with 4, 12 and 24 mM of KNO3 in 21 healthy subjects. These 395

authors found a 7-fold increase in peak plasma [NO3-] following 4 mM KNO3, a 27-fold increase 396

following 12 mM KNO3, and a 35-fold increase following 24 mM KNO3 (Kapil et al. 2010). The 397

peak plasma [NO3-] occurred 3 h post-ingestion. In a similar manner, these authors found a 1.3-398

fold increase in peak plasma [NO2-] following 3 mM KNO3, a 2-fold increase following 12 mM 399

KNO3, and a 4-fold increase following 24 mM KNO3. The peak plasma [NO2-] occurred 2.5 h 400

post-ingestion. Interestingly, this dose-response relationship translated to dose-dependent 401

reductions in systolic blood pressure (SBP) and diastolic blood pressure (DBP). When 4 mM 402

6

KNO3 was administered, the peak reduction in SBP was 2 mmHg and the peak reduction in DBP 403

was 4 mmHg. When 12 mM KNO3 was administered, the peak reduction in SBP was 6 mmHg 404

and the peak reduction in DBP was 4 mmHg. Lastly, when 24 mM KNO3 was administered, the 405

peak reduction in SBP was 6 mmHg and the peak reduction in DBP was 5 mmHg (Kapil et al. 406

2010). The results from this study demonstrate that plasma NO3- and NO2- increase in a dose-407

dependent manner which correlates with reductions in SBP and DBP. 408

Wylie et al. (2013) investigated the dose-response relationship of 4.2, 8.4, and 16.8 mM 409

of dietary NO3- administered as an acute dose of BRJ in 10 recreationally active males. These 410

authors found an 8-fold increase in peak plasma [NO3-] following 4.2 mM NO3-, a 12-fold 411

increase following 8.4 mM NO3-, and a 24-fold increase following 16.8 mM NO3- (Figure 2). 412

The peak plasma [NO3-] occurred 1 h post-ingestion following 4.2 and 8.4 mM dietary NO3- and 413

2 h post-ingestion for 16.8 mM dietary NO3-. Similarly, these authors found a 3.3-fold increase 414

in peak plasma [NO2-] following 4.2 mM NO3-, a 5-fold increase following 8.4 mM NO3-, and a 415

7.8-fold increase following 16.8 mM NO3-. The peak plasma [NO2-] occurred 2 h post-ingestion 416

following 4.2 and 8.4 mM dietary NO3- and 4 h post-ingestion for 16.8 mM dietary NO3-. 417

Similar to the results presented by Kapil et al. (2010), these authors found a dose-418

dependent relationship in the reductions of SBP, DBP, and mean arterial pressure (MAP). An 419

acute dose of 4.2 mM NO3- resulted in peak reductions of 4 mmHg in SBP, 1 mmHg in DBP, 420

and 2 mmHg in MAP. When an acute 8.4 mM NO3- dose was administered, peak reductions 421

were 8 mmHg in SBP, 3 mmHg in DBP, and 4 mmHg in MAP. Lastly, when an acute 16.8 mM 422

NO3- dose was administered, peak reductions were 10 mmHg in SBP, 4 mmHg in DBP, and peak 423

5 mmHg in MAP (Wylie et al. 2013). 424

7

The same authors also investigated the dose-response relationship with regard to the 425

reductions in baseline and end-exercise VO2 during moderate (~105 watts (W)) and severe 426

intensity (~300 W) exercise to exhaustion. There was no change in baseline VO2 with moderate 427

or severe intensity exercise. There was no change in end-exercise VO2 with an acute dose of 4.2 428

mM NO3-, however when an acute dose of 8.4 mM NO3- was administered there was a 2% 429

reduction in end-exercise VO2 during moderate intensity exercise, and the acute dose of 16.8 430

mM resulted in a 3% reduction in end-exercise VO2 with moderate intensity exercise (Wylie et 431

al. 2013). There was no effect of any dose of BRJ supplementation on end-exercise VO2 during 432

the severe exercise bout. Interestingly, an acute dose of 8.4 mM NO3- resulted in a significant 433

improvement in time to exhaustion during severe intensity exercise compared to placebo (PLA) 434

(8.4 mM: 570 + 153 vs. PLA: 498 + 113 s). Time to exhaustion was also significantly improved 435

with an acute dose of 16.8 mM NO3- (552 + 117 s) compared to PLA (493 + 114 s). The results 436

from this study indicate that an acute dose of at least 8.4 mM NO3- is required to elicit positive 437

effects on BP, the O2 cost of exercise, and to improve time to exhaustion. 438

439

8

440

Figure 2: The pharmacokinetic profile and dose response relationship of 4.2, 8.4 and 16.8 mM 441 dietary nitrate (NO3-) on plasma NO3- and nitrite (NO2-). Filled circles, control treatment; filled 442 triangles, 4.2 mM; filled squares, 8.4 mM; and filled diamonds, 16.8 mM (Wylie et al. 2013). 443 444 Acute vs. Chronic Supplementation: Plasma Levels, VO2 and Performance 445

Few studies have directly compared the effects of different durations of dietary NO3- 446

supplementation on blood parameters, health, and performance (Vanhatalo et al. 2010; Fulford et 447

al. 2013; Wylie et al. 2016). Vanhatalo et al. (2010) investigated the effects of acute and chronic 448

BRJ supplementation on BP and exercise performance in 8 healthy participants (5 males). These 449

authors found that mean plasma [NO2-] measured 2.5 h post-ingestion increased by 36% 450

following an acute dose of 5.2 mM BRJ, by 59% following 12 days of supplementation, and by 451

46% following 15 days of supplementation. SBP was lowered by ~5 mmHg with BRJ 452

supplementation at all time points, DBP was lowered by ~4 mmHg, and MAP was lowered by 453

~5 mmHg. Furthermore, moderate intensity (~90 W) end-exercise VO2 was consistently reduced 454

by ~5% following acute supplementation, 5 days of supplementation and 15 days of 455

9

supplementation. Fulford et al. (2013) explored the effects of dietary NO3- on skeletal muscle 456

force production both acutely and chronically in 8 healthy males. These authors demonstrated 457

that plasma [NO2-] measured at 2.5 h post-ingestion increased by a similar amount following an 458

acute dose of 10.2 mM NO3-, and after 5 and 15 days of supplementation. Interestingly, there 459

was no impact of acute or chronic BRJ supplementation on skeletal muscle contractile force 460

production (Fulford et al. 2013). Unfortunately, these authors did not measure VO2. 461

Wylie et al. (2016) reported the pharmacokinetic profiles and exercise effects associated 462

with different durations and doses of dietary NO3- supplementation in 34 recreationally active 463

subjects (19 males). These authors found that with daily doses of both 3 and 6 mM of dietary 464

NO3-, there was no significant difference within conditions for plasma [NO3-] or [NO2-] 465

following an acute dose, 7 days of supplementation or 30 days of supplementation. However, 466

plasma [NO3-] and [NO2-] were significantly greater in the 6 mM condition compared to the 3 467

mM condition, where plasma [NO3-] increased by 313% in the 3 mM dietary NO3- condition and 468

by 867% in the 6 mM dietary NO3- condition (Wylie et al. 2016). Plasma [NO2-] increased by 469

165% in the 3 mM condition and 579% in the 6 mM condition (Wylie et al. 2016). There was no 470

effect of the 3 mM condition on end-exercise VO2 during moderate intensity exercise (~90 W). 471

Conversely, the rise in plasma [NO2-] in the 6 mM condition was associated with a 3% reduction 472

in end-exercise VO2 during moderate intensity exercise. These authors also investigated the 473

effects of chronic supplementation without an acute dose administered 2 h prior to blood 474

sampling and exercise testing. Without the acute dose, plasma [NO3-] returned to baseline levels 475

by 24 h post-ingestion in the 3 mM condition and declined significantly in the 6 mM condition. 476

However, plasma [NO3-] was still significantly elevated above baseline in the 6 mM condition 24 477

h following the last dose of BRJ. Without the acute dose, plasma [NO2-] returned to baseline by 478

10

24 h post-ingestion in the 3 and 6 mM conditions. Similar to the chronic supplementation with 479

the acute dose administered prior to testing, there was no change in end-exercise VO2 in the 3 480

mM condition. Interestingly, when the dose was 6 mM per day the 3% reduction in VO2 was 481

conserved despite no acute dose administered 2 h prior to testing. 482

From these studies, it appears that subjects do not develop a tolerance to dietary NO3- 483

with up to 30 days of supplementation, and the effects on blood parameters, health, and exercise 484

performance are similar both acutely and chronically. Further corroborating these findings, Kelly 485

et al. (2013) had subjects visit the laboratory over 5 consecutive days during days 3 through 7 of 486

dietary NO3- supplementation and reported no difference in plasma [NO2-] measured 2.5 h post-487

ingestion across the 5 days, with BRJ consistently improving time to exhaustion to the same 488

extent (~ 13%) during severe intensity exercise tests (Kelly et al. 2013). 489

The Effects of BRJ on Exercise Economy and Athletic Performance 490

Moderate Intensity Submaximal Exercise Economy 491

For the purpose of this thesis, exercise economy is defined as a reduction in the O2 cost of 492

exercise at a fixed workload. A basic tenet of human exercise physiology states that the O2 cost 493

of submaximal exercise at a given work rate is fixed regardless of age, health and fitness status 494

(Poole and Richardson 1997). Furthermore, this tenet suggests that the O2 cost of submaximal 495

exercise is unaltered by physical, nutritional or pharmacological interventions (Poole and 496

Richardson 1997). It has been demonstrated that O2 uptake increases linearly as a function of 497

work rate, where O2 consumption is ~9 to 11 mL O2/W/min during moderate intensity exercise 498

(Poole and Richardson 1997). BRJ supplementation challenges this dogma by consistently 499

demonstrating reductions, typically between 3 to 5%, in the O2 cost of moderate intensity 500

submaximal exercise in recreationally active males as reviewed by Affourtit et al. 2015. 501

11

Moderate intensity exercise is characterized by low production and adequate clearance of 502

blood lactate, and therefore is generally classified as exercise that falls below the lactate 503

threshold (Xu and Rhodes 1999). The lactate threshold is defined as the blood lactate 504

concentration at which lactate production exceeds its clearance and accumulation occurs. The 505

onset of blood lactate accumulation occurs at an arterial lactate concentration of 4 mM/L and 506

typically occurs around 75% maximal oxygen uptake (VO2max) (Ghosh 2004). The VO2 kinetics 507

associated with moderate intensity exercise can be divided into three major components. Phase I 508

is depicted by the early and rapid rise in VO2 that occurs in the first 15 to 25 s of exercise, and 509

this has been linked to the rise in cardiac output and blood flow at the onset of exercise (Xu and 510

Rhodes 1999) (Figure 3). Phase II is represented by an exponential rise in VO2 towards steady 511

state and reflects shifts in skeletal muscle metabolism and substrate utilization. Phase III is 512

achieved after ~3 min of exercise and is defined as steady state VO2, which has been described 513

previously as ~9 to 11 mL O2/W/min and is indicated by a heavy reliance on aerobic 514

metabolism. BRJ supplementation has been shown to influence VO2 kinetics by decreasing the 515

primary VO2 amplitude and ultimately decreasing end-exercise VO2 in recreationally active 516

groups composed of predominantly male subjects (Bailey et al. 2009; Vanhatalo et al. 2010; 517

Lansley et al. 2011) (Figure 3). 518

Interestingly, these reductions in the O2 cost of exercise are present merely 2.5 h 519

following an acute dose of dietary or inorganic NO3- and persist for up to 30 days of chronic 520

supplementation. Furthermore, these effects are seen with doses ranging from 5.2 mM to 16.8 521

mM dietary or inorganic NO3-. BRJ supplementation has shown to be effective at reducing the 522

O2 cost of exercise at intensities ranging from 45 to 75% VO2peak and these reductions have been 523

elicited across a range of exercise modalities including walking (Lansley et al. 2011), running 524

12

(Lansley et al. 2011), cycling (Larsen et al. 2007; 2010; 2011; Bailey et al. 2009; Vanhatalo et al. 525

2010; Reink et al. 2015; Wylie et al. 2013; Whitfield et al. 2016) and knee extension exercise 526

(Bailey et al. 2010). 527

528 Figure 3: An illustration of the effects of beetroot juice (nitrate) supplementation on oxygen 529 uptake (VO2) kinetics from rest to moderate intensity exercise in recreationally active males. 530 Nitrate significantly reduced the oxygen cost of exercise by reducing the primary amplitude of 531 VO2 kinetics (Lansley et al. 2011). 532 533 Severe and Maximal Intensity Exercise Economy 534

Severe intensity exercise exceeds the lactate threshold. Although phase II kinetics still 535

exist in this exercise domain, VO2 kinetics are complicated by the VO2 slow component that 536

develops after a few minutes at this intensity (Xu and Rhodes 1999). During severe intensity 537

exercise, VO2 steady state (phase III) is not achieved and the slow component continues to 538

increase until task failure (Xu and Rhodes 1999). Two studies have investigated VO2 kinetics 539

during severe intensity exercise (Bailey et al. 2009; Breese et al. 2013). These groups define 540

severe intensity exercise as 70% of the difference between the gas exchange threshold (GET) and 541

peak oxygen consumption (VO2peak) (Bailey et al. 2009, ~275 W; Breese et al. 2013, ~215 W). 542

13

Bailey et al. (2009) found a significant reduction in the VO2 slow component amplitude with 6 543

days of BRJ supplementation, which translated to a 16% improvement in time to exhaustion in 8 544

healthy men. These authors also found a significantly decreased primary VO2 amplitude 545

following BRJ supplementation compared to PLA (Bailey et al. 2009). Conversely, Breese et al. 546

(2013) found no significant difference in the VO2 slow component amplitude or the primary VO2 547

amplitude following 6 days of BRJ supplementation compared to PLA in 9 healthy men. These 548

discrepancies may exist because of differing exercise protocols in which Bailey et al. (2009) 549

transitioned from very low intensity exercise to severe intensity exercise, whereas Breese et al. 550

(2013) transitioned from moderate intensity exercise to severe intensity exercise. A greater body 551

of literature is required to elucidate the VO2 kinetic profiles associated with BRJ 552

supplementation and severe intensity exercise. 553

To date, three studies have investigated the effects of BRJ supplementation on VO2max 554

(Larsen et al. 2007; 2010; Vanhatalo et al. 2010). The initial study by Larsen et al. (2007) 555

demonstrated no significant effect of 3 days of NaNO3 supplementation on VO2max in 9 well-556

trained males. However, in 2010 these authors found a significant reduction in VO2max following 557

3 days of NaNO3 supplementation in 9 healthy volunteers (7 males) (Larsen et al. 2010). A 558

reduced VO2max would likely indicate impaired exercise performance. However, Vanhatalo et al. 559

(2010) demonstrated an increase in VO2max following 15 days of BRJ supplementation in 8 560

recreationally active individuals (5 males). It is difficult to reconcile the array of outcomes seen 561

across these studies and a greater body of research is required to determine if dietary NO3- 562

substantially impacts maximal exercise performance. 563

564

565

14

Time Trial Performance 566

Time trial performance is typically measured as a set distance, set amount of work, or set 567

time allocated for exercise performance. Time trials hold the greatest real-world validity when 568

measuring exercise performance in athletes and are the most reliable and reproducible protocols 569

(Currell and Jeukendrup 2008). There are no distinguishable differences in variability between 570

time trial exercise modalities where the coefficient of variation (CV) is below 5% for cycling, 571

running, and rowing (Currell and Jeukendrup 2008). 572

Aerobic Cycling Time Trial Performance 573

A few studies have demonstrated an improvement in aerobic cycling time trial 574

performance following dietary NO3- supplementation. Although some of these studies did not 575

achieve statistical significance, they fall below the smallest worthwhile change for cycling time 576

trial performance in elite cyclists as described by Hopkins (2004). This statistical inference 577

suggests that the average CV for cycling time trials is ~ 0.6% for trained cyclists, and any change 578

that exceeds this may be deemed worthwhile (Paton and Hopkins 2006). Lansley et al. (2011) 579

found a 2.8% improvement in 4 km time trial performance, and a 2.7% improvement in 16.1 km 580

time trial performance following an acute dose of BRJ in 9 competitive male cyclists. Wilkerson 581

et al. (2012) found a 0.8% improvement on a 50 mile time trial following an acute dose of BRJ 582

in 8 trained male cyclists, and Cermak et al. (2012) found a 1.3% improvement in 10 km time 583

trial performance following 6 days of BRJ supplementation in 13 trained male cyclists and 584

triathletes. Lastly, Lee et al. (2015) found a 2.1% improvement in 20.15 km time trial 585

performance following acute supplementation with a NO containing lozenge in 16 trained 586

cyclists (8 males). All of these studies utilized a single-blinded testing protocol for the subjects 587

or a double-blinded testing protocol. However, none of these studies administered a post-testing 588

15

questionnaire to confirm if the subjects were successfully blinded to the supplementation 589

regimes. 590

Interestingly, there are also a number of studies that demonstrated no significant effect on 591

aerobic cycling time trial performance following dietary NO3- supplementation (Cermak et al. 592

2012; Lane et al. 2013; Christensen et al. 2013; Glaister et al. 2015; McQuillan et al. 2016; 593

Nyakayiru et al. 2016; Callahan et al. 2017). It is important to note that although dietary NO3- 594

supplementation may not elicit a beneficial effect on time trial performance, it has not shown to 595

be detrimental to athletic performance. Furthermore, it is important to consider that these time 596

trial studies were conducted in trained athletes, and their training status may contribute to the 597

controversial findings on athletic performance following dietary NO3- supplementation. This will 598

be addressed in a subsequent section. 599

Aerobic Running Time Trial Performance 600

There is a limited body of evidence investigating the effects of dietary NO3- 601

supplementation on aerobic running time trial performance and the outcomes are equivocal 602

(Murphy et al. 2012; Peacock et al. 2012; Boorsma et al. 2014). Murphy et al. (2012) found a 603

significant improvement in 5 km time trial performance, where running velocity was increased 604

by 5% following an acute dose of BRJ in 11 healthy individuals (5 males). Conversely, Peacock 605

et al. (2012) found no effect of an acute dose of KNO3 on 5 km running time trial performance in 606

10 elite male cross-country skiers. Similarly, Boorsma et al. (2014) found no significant effect of 607

acute or chronic BRJ supplementation on 1500 m running performance in 10 elite male distance 608

runners. The smallest worthwhile change for elite athletes in these events is a 1.1% improvement 609

in performance as described by Hopkins (2004). Neither Peacock et al. (2012) or Boorsma et al. 610

(2014) found performance benefits greater than these parameters. Again, training status may 611

16

have confounded the performance benefits associated with dietary NO3- supplementation, as 612

Peacock et al. (2012) and Boorsma et al. (2014) recruited elite runners whereas, Murphy et al. 613

(2012) recruited recreationally active individuals. 614

Water Sport Time Trial Performance 615

There is some evidence to suggest that BRJ may improve performance in water sports 616

such as rowing, kayaking, and swimming. The CV for elite rowing time trial performance is 617

noted to be ~ 0.4%, therefore any improvement greater than the CV is predicted to be a 618

worthwhile improvement (Hopkins 2004). Hoon et al. (2012) showed that acute BRJ 619

supplementation was possibly beneficial (~2 s improvement) to 2000 m rowing performance in 620

10 highly trained male rowers. Bond et al. (2012) demonstrated that 6 days of BRJ 621

supplementation was likely beneficial during 6 x 500 m rowing sprints, where the benefit was 622

almost certain in sprints 4 through 6. The CV for elite kayaking time trial performance is ~ 0.6% 623

(Hopkins 2004) and Peeling et al. (2015) found a 0.7% improvement in the distance covered 624

during a 4 min kayaking test in 6 national level male kayakers and found a 1.7% improvement in 625

500 m kayaking time trial performance in 5 international level female kayakers. However, 626

Muggeridge et al. (2013) saw no difference in 1 km kayaking time trial performance despite a 627

significantly reduced submaximal VO2 (3.1%) when kayaking for 15 min at 60% maximal 628

workload (Wmax) following an acute dose of BRJ in 8 trained male kayakers. Pospieszna et al. 629

(2016) investigated the effects of 8 days of BRJ supplementation on 6 x 50 m sprint swimming 630

performance, and 800 m endurance swimming performance in 11 female collegiate swimmers. 631

These authors noted a statistically significant improvement in sprints 4 through 6 following BRJ 632

supplementation as well as improvements in 800 m time trial performance. 633

634

17

Time to Exhaustion 635

A number of studies have reported improved time to exhaustion following BRJ 636

supplementation in healthy, recreationally active males. These improvements have been reported 637

with both acute supplementation and chronic supplementation lasting up to 15 days. 638

Furthermore, time to exhaustion has been enhanced across a range of exercise modalities 639

including: cycling (Bailey et al. 2009; Larsen et al. 2010; Wylie et al. 2013; Kelly et al. 2013; 640

Thompson et al. 2014; Bailey et al. 2015), running (Lansley et al. 2011), and knee extension 641

exercise (Bailey et al. 2010; Hoon et al. 2015). The reported improvements have been between 7 642

and 20%. 643

Sprint and Team Sport Performance 644

To date, only two studies have investigated the effects of BRJ supplementation on sprint 645

cycling performance. Rimer et al. (2016) found that acute BRJ supplementation increased peak 646

power during 4 sets of 3 – 4 s sprints, however there was no benefit of BRJ supplementation on 647

peak or mean power during a 30 second Wingate in 13 competitively trained athletes from an 648

array of sports (11 males). Conversely, after an acute dose of BRJ, Dominguez et al. (2017) 649

noted a significant increase in peak power during a 30 s Wingate, but no difference in mean 650

power in 15 recreationally active males. Since little literature exists, the results are equivocal and 651

require further investigation. 652

Due to the complexity and multifaceted nature of team sports, it is difficult to quantify 653

team sport performance in a controlled setting. One commonly employed method to bypass the 654

complexity, uncertainty, and spontaneity of stop-and-go sporting events is to create a simulated 655

environment that closely mimics gameplay. Stop-and-go team sports are typically characterized 656

by intermittent and repeated sprints, and protocols such as the running Loughborough 657

18

Intermittent Shuttle Test or the Yo-Yo Intermittent Recovery Test have been highly correlated 658

with movement patterns utilized in team sports (Currell and Jeukendrup 2008). There is mixed 659

support for the benefit of BRJ supplementation on team sport performance as assessed by 660

repeated sprint efforts. It has been shown that acute (Wylie et al. 2013) and chronic (Thompson 661

et al. 2016) BRJ supplementation can increase the distance covered during the Yo-Yo 662

Intermittent Recovery Test by ~ 4% in male recreational team-sport athletes. Another study 663

demonstrated a significant increase in mean power during 24 sets of 6 s sprints, however there 664

was no improvement during 7 sets of 30 s sprints, or 6 sets of 60 s sprints with 5 days of BRJ 665

supplementation in 34 male recreationally active team-sport athletes (Wylie et al. 2016). On the 666

contrary, Buck et al. (2015) failed to demonstrate an improvement in repeated sprint 667

performance with acute BRJ supplementation in 13 female amateur team-sport athletes. 668

Although it appears that BRJ supplementation may be beneficial for team sport performance, a 669

greater body of literature is required to validate and confirm these findings. 670

Strength Performance 671

There is minimal research published regarding the effects of BRJ supplementation on 672

strength performance. Mosher et al. (2016) demonstrated that acute supplementation with BRJ 673

improved the number of repetitions to failure and consequently the total weight lifted at failure 674

during bench press performed at 60% of 1 repetition maximum in 12 recreationally active males. 675

Conversely, Flanagan et al. (2016) found no significant effect of 3 days of BRJ supplementation 676

on the maximum number of repetitions performed at 60, 70, 80 or 90% 1 repetition maximum in 677

14 resistance-trained males. 678

679

680

19

Performance During Hypoxia 681

Exercise in simulated hypoxic environments is characterized by a reduced inspired O2 682

concentration, ultimately resulting in reduced arterial O2 content, reduced oxidative capacity of 683

contracting muscles, and impaired exercise tolerance (Vanhatalo et al. 2011; Kelly et al. 2014). 684

The reduction in inspired O2 can compromise the L-arginine pathway for NO production since it 685

relies on O2 as a substrate (Lundberg et al. 2010). Therefore, BRJ supplementation may be 686

important in hypoxic conditions because it augments the sequential reduction of NO3- to NO 687

which does not rely on O2. Most of the literature studying BRJ supplementation in hypoxia (15% 688

O2) demonstrates a profound 6 – 8% decrease in the O2 cost of exercise during moderate 689

intensity cycling (~135 – 200W) (Kelly et al. 2014; Muggeridge et al. 2014) (Figure 4), and 690

significant improvements in time to task failure (Vanhatalo et al. 2011; Kelly et al. 2014) and TT 691

performance (Muggeridge et al. 2014). Interestingly, Vanhatalo et al. (2011) found that BRJ 692

supplementation significantly attenuated the rate of metabolic perturbation associated with high 693

intensity knee extension exercise in a hypoxic environment (~15% O2), and restored exercise 694

tolerance to normoxic conditions in 9 recreationally active subjects (7 males). Specifically, after 695

60 s and 120 s of exercise, phosphocreatine (PCr) had fallen to a greater extent in the PLA 696

condition compared to the BRJ condition. Consequently, inorganic phosphate (Pi) accumulation 697

and pH reduction were greater in the PLA condition compared to the BRJ condition, ultimately 698

indicating a greater reliance on aerobic metabolism following BRJ supplementation in hypoxic 699

conditions. There was no difference in these parameters between the hypoxic BRJ condition and 700

the control normoxic condition indicating a scenario by which BRJ restored exercise tolerance. 701

702

20

703 Figure 4: An illustration of the effects of beetroot juice (BRJ) supplementation (nitrate) on 704 oxygen uptake kinetics during moderate intensity exercise (~135 W) in simulated altitude (15% 705 oxygen (O2)) in recreationally active males. There was an ~8% reduction in the O2 cost of 706 exercise with BRJ during hypoxia compared to a 3% reduction in the O2 cost of exercise with 707 BRJ during normoxia. (Kelly et al. 2014). 708 709 Conclusion 710

BRJ supplementation has been shown to elicit profound effects on exercise economy and 711

athletic performance. Specifically, BRJ supplementation has been shown to reduce the O2 cost of 712

moderate intensity exercise and improve time to exhaustion. These findings are exacerbated in 713

hypoxic situations. The effects of BRJ supplementation on time trial, sprint, team sport, and 714

strength performance are equivocal and often confounded by a lack of literature in the field. 715

Alternatively, it is often suggested that an athlete’s training status may impact their response to 716

dietary NO3- supplementation. This will be addressed in the following section. 717

Individual Responses to BRJ Supplementation 718

There is inherently individual variability in the physiological responses to nutritional 719

interventions, and the likelihood that an individual is going to respond positively to a nutritional 720

intervention is influenced by a multitude of factors. Furthermore, the magnitude of responses is 721

influenced by these very same factors and can be visualized as a continuum. When considering 722

21

the factors that influence the response to BRJ supplementation, it is important to consider 723

differences in the fitness status, dietary habits, and digestive pathway of the subjects. A “non-724

responder” is identified as an individual who consistently does not display a reduction in the O2 725

cost of exercise following BRJ supplementation. Conversely, a “responder” is an individual who 726

consistently demonstrates a reduction in the O2 cost of exercise that is greater than the coefficient 727

of variance for the measurement. 728

Interestingly, genetics may also influence an individual’s physiological responses to NO. 729

A recent study by Emdin et al. (2017) demonstrated that certain individuals may have a genetic 730

predisposition for enhanced NO signalling. This was associated with a reduced risk of coronary 731

heart disease and stroke. Interestingly, individuals who had rare genetic variants that inactivated 732

certain NO signalling genes demonstrated significantly elevated blood pressure and a 3-fold 733

increased risk of coronary heart disease (Emdin et al. 2017). Future studies should investigate the 734

possibility that individuals who have enhanced endogenous NO signalling may be less likely to 735

respond to dietary NO3- supplementation. 736

Fitness Status 737

There is substantial evidence to suggest that highly trained individuals may not respond 738

to dietary NO3- supplementation and therefore do not demonstrate a reduced O2 cost of 739

submaximal exercise (Peacock et al. 2012; Wilkerson et al. 2012; Christensen et al. 2013; 740

Boorsma et al. 2014; Lee et al. 2015; McQuillan et al. 2016; Nyakayiru et al. 2016). A study by 741

Porcelli et al. (2015) investigated the effects of chronic NaNO3 supplementation on plasma 742

[NO3-] and [NO2-], the O2 cost of exercise and time trial performance in 21 males with either 743

low, moderate, or high aerobic fitness. It was discovered that individuals with high aerobic 744

fitness had higher baseline plasma [NO3-] compared to individuals with low or moderate aerobic 745

22

fitness. Furthermore, the individuals with low or moderate aerobic fitness demonstrated the 746

greatest rises in both relative and absolute plasma [NO3-] and [NO2-] following chronic 747

supplementation with NaNO3. These authors identified that individuals with low and moderate 748

aerobic fitness elicited a 9.8% and 7.3% reduction in the O2 cost of exercise at 80% GET, 749

respectively. Interestingly, chronic NaNO3 supplementation also resulted in a significant 750

improvement in 3 km running time trial performance for individuals with low and moderate 751

aerobic fitness. However, there was no difference in the O2 cost of exercise or 3 km running time 752

trial performance for individuals with high aerobic fitness. It is possible that the individuals with 753

low and moderate aerobic fitness may experience performance benefits with dietary NO3- due to 754

the dramatic elevation in plasma [NO2-] following supplementation compared to individuals with 755

high aerobic fitness. Similar results were obtained by Carriker et al. (2016) who found a reduced 756

O2 cost of exercise at 45% (~90 W) and 60% (~150 W) VO2max in 5 males with low aerobic 757

fitness (VO2max, 42.4 + 3.2 mL/kg/min). However, these results were not found in 6 males with 758

high aerobic fitness levels (VO2max, 60.1 + 4.6 mL/kg/min) (Carriker et al. 2016). 759

Jonvik et al. (2016) collected diet records from hundreds of elite athletes to assess their 760

habitual dietary NO3- intake. These authors hypothesized that elite athletes may have a blunted 761

response to dietary NO3- supplementation because they may already have diets rich in these 762

foods. However, it was discovered that the average dietary NO3- intake was only 106 mg/d, 763

where a typical dose of BRJ is at least 400 mg of dietary NO3-. Furthermore, only 10% of the 764

athletes consumed greater than 400 mg/d of dietary NO3-. This suggests that there are other 765

physiological mechanisms at play with elite athletes and their blunted response to dietary NO3- 766

supplementation. Interestingly, Jonvik et al. (2016) reported that female athletes typically 767

ingested a larger absolute amount of dietary NO3- per day than male athletes, despite having 768

23

lower total energy intake. This was attributed to a greater consumption of vegetables by female 769

athletes. 770

Diet 771

It is possible that individuals who habitually consume high levels of dietary NO3- may not 772

respond to BRJ supplementation since they already express elevated plasma [NO3-] and [NO2-]. 773

However, it is expected that these differences would manifest as variations in individual plasma 774

[NO3-] and [NO2-]. It has been shown that traditional Japanese diets are high in dietary NO3-, and 775

adherence to this diet is associated with lower SBP and DBP (Gee and Ahluwalia 2016). 776

Furthermore, when the same Japanese population was placed on a western diet they experienced 777

significant increases in SBP and DBP (Gee and Ahluwalia 2016). It has been suggested that the 778

high dietary NO3- in the Japanese diet may be a major contributor to the population’s longevity 779

and low rates of cardiovascular disease (Gee and Ahluwalia 2016). Similarly, other authors argue 780

that individuals who follow the Dietary Approaches to Stop Hypertension (DASH) diet 781

experience reductions in blood pressure, which is partially attributed to the high leafy green, and 782

consequently high dietary NO3- content, of this diet (Ashworth et al. 2015). 783

Digestive Pathway 784

It is expected that there is interindividual variability in the way we absorb, process, and 785

store nutrients. When analyzing the potential variability in the responses to BRJ 786

supplementation, it is important to consider interindividual differences that may exist in the 787

enterosalivary circulation. First, it is possible that there are differences in the amount of NO3- 788

that is absorbed from the stomach and small intestines. This may mean that there is 789

interindividual variability in the fraction of dietary NO3- that is excreted from the body. Second, 790

the rate of and magnitude of NO3- concentration into the salivary glands may differ between 791

24

individuals. Next, there are likely differences in the amount of and activity of the bacteria that 792

reduce NO3- to NO2- inside the mouth. Lastly, there may be interindividual differences in the 793

storage, conversion and degradation of dietary NO3- that is absorbed as NO2-. There is currently 794

no literature focusing on these aspects as they are difficult to quantify and measure. However, it 795

is expected that these differences would manifest as variations in individual plasma [NO3-] and 796

[NO2-]. Interestingly, Kapil et al. (2010) found that plasma [NO2-] rose 2-fold higher in female 797

subjects (n = 12) compared to males (n = 8) following an acute dose of KNO3. This trend still 798

existed when plasma [NO2-] was normalized to body weight. These authors suggest that sex 799

differences may exist in the way nitrate loads are processed. 800

BRJ Improves Exercise Economy and Performance – Potential Mechanisms of Action 801

Are We Sure It’s Nitrate? 802

The early BRJ studies were performed before the development of a NO3--free PLA which 803

is manufactured using ion resin exchange (Lansley et al. 2011). This PLA was first introduced in 804

the BRJ literature by Lansley et al. in 2011. Up until that point, it was difficult to ascertain 805

whether the beneficial effects of BRJ supplementation were strictly from its dietary NO3- content 806

or if there were other bioactive properties in the beverage. It is now abundantly clear that the 807

main bioactive in BRJ is dietary NO3- since the beneficial effects are only reported with the NO3-808

-rich beverage compared to the NO3--free placebo. However, some authors continue to argue that 809

other bioactives within BRJ are a major contributor to its positive biological effects. A number of 810

recent publications have attributed the effects of BRJ supplementation to its high antioxidant 811

status. BRJ is high in betalain pigment, an antioxidant that gives the vegetable it’s deep red 812

colour (Clifford et al. 2016b). Specifically, BRJ is high in betanin, a subclass of the betalain 813

pigment. Clifford et al. (2016a; 2016b; 2017) have continually explored the antioxidant effects of 814

25

BRJ supplementation on muscle damage, recovery and performance in recreationally active 815

males. They have consistently shown significant reductions in muscle soreness as well as 816

attenuated performance decrements assessed using a countermovement jump following fatiguing 817

exercise with 3 days of BRJ supplementation (Clifford et al. 2016a; 2016b). However, these 818

authors have failed to show a benefit with respect to running performance or changes in 819

biomarkers of inflammation (Clifford et al. 2016b; 2017a). Furthermore, despite the high betanin 820

content of BRJ, these authors failed to demonstrate an increase in plasma betanin over an 8 h 821

period following acute BRJ supplementation suggesting that this antioxidant may not play an 822

ergogenic role (Clifford et al. 2017b). A major limitation of these studies is that they cannot 823

differentiate the potential anti-inflammatory effects of NO3- compared to betalain or betanin 824

since a NO3--free PLA was not utilized. However, in a separate set of experiments, Hoorebeke et 825

al. (2016) demonstrated that concentrated betalain supplementation improved perceived exertion 826

during submaximal treadmill running and improved 5 km time trial performance in 15 827

recreationally active males. Similarly, Montenegro et al. (2017) found that concentrated betalain 828

supplementation improved performance on a 10 km time trial, and a 5 km time trial performed 829

the next day in 22 triathletes (9 males). It is important to note, that neither of these studies 830

demonstrated a reduction in the O2 cost of exercise following supplementation suggesting 831

betalain is not responsible for this physiological response to BRJ supplementation (Hoorebeke et 832

al. 2016; Montenegro et al. 2017). Cumulatively, although it is important to recognize that there 833

are other bioactives in BRJ that may contribute to its beneficial effects, the antioxidant properties 834

associated with betalain fail to explain the consistent reduction in the O2 cost of exercise seen 835

with dietary NO3- interventions. 836

26

BRJ supplementation may improve exercise performance through a variety of 837

mechanisms. The potential mechanisms for the reduced O2 cost of exercise range from improved 838

O2 delivery and waste removal to improved contractile or mitochondrial efficiency. It has also 839

been proposed that BRJ may improve exercise performance by attenuating skeletal muscle 840

damage or facilitating mitochondrial biogenesis. 841

Improved Blood Flow 842

NO is renowned for its role as a potent vasodilator. This occurs through the interaction of 843

NO with guanylyl cyclase (Gc) which ultimately increases cyclic guanosine monophosphate 844

(cGMP) and facilitates the removal of Ca2+ from the smooth muscle cell resulting in relaxation. 845

Furthermore, NO has also been shown to improve skeletal muscle blood flow. Ferguson et al. 846

(2013a) performed a series of studies utilizing rats and BRJ supplementation. The first study 847

demonstrated a significant increase in hindlimb skeletal muscle blood flow and vascular 848

conductance during treadmill running in rats undergoing 5 days of BRJ supplementation. The 849

second study demonstrated that 5 days of BRJ supplementation attenuated the decline in O2 850

delivery/O2 utilization that occurs between contractions, meaning that the drive for O2 was 851

elevated in the periods between contractions providing enhanced delivery (Ferguson et al. 852

2013b). In humans, Richards et al. (2017) found increased forearm blood flow following acute 853

BRJ supplementation during a dynamic handgrip task in 18 healthy subjects (8 males). Although 854

this does not explain the reduced O2 cost of exercise, it may be an important factor related to the 855

performance benefits of BRJ since this may augment O2 delivery as well as CO2 and waste 856

removal. Future research needs to investigate the effects of BRJ supplementation with direct 857

measures of blood flow in human subjects. 858

859

27

Improved Mitochondrial Function 860

Reduction in the O2 Cost of Mitochondrial Adenosine Triphosphate (ATP) Resynthesis 861

The electron transport chain (ETC) is comprised of a series of complexes that use 862

electrons from NADH and FADH2 to facilitate the movement of protons (hydrogen (H+) ions) 863

from the mitochondrial matrix to the intermembrane space creating an electrochemical gradient 864

that drives the synthesis of ATP via mitochondrial membrane potential (Farrell et al. 2012). 865

Importantly, the final electron acceptor in the ETC is O2, and ATP is resynthesized from 866

adenosine diphosphate (ADP) and inorganic phosphate via the enzyme ATP synthase (Farrell et 867

al. 2012). The efficiency of ATP resynthesis depends on the rate of proton leak and slippage 868

(Farrell et al. 2012). Proton leak occurs when H+ ions are utilized via alternative pathways that 869

do not contribute to the generation of ATP such as uncoupling protein 3 (UCP-3). Slippage 870

occurs when electrons are moved without the pumping of protons, ultimately resulting in a 871

decreased membrane potential to drive ATP resynthesis (Farrell et al. 2012). The efficiency of 872

mitochondrial respiration is often expressed as the phosphate/O2 (P/O) ratio and is defined as the 873

rate of ATP synthesis divided by the rate of O2 consumption (Larsen et al. 2011). 874

It has been proposed that dietary NO3- supplementation can improve the efficiency of 875

ATP resynthesis by reducing proton leak and slippage (Clerc et al. 2007; Larsen et al. 2011). 876

Larsen et al. (2011) associated the reduced O2 cost of exercise following 3 days of NaNO3 877

supplementation with an improved P/O ratio. Specifically, these authors found a significant 878

reduction in state 4 respiration, which is characterized by availability of respiratory substrate but 879

not ADP and estimates the back leakage of protons through the inner mitochondrial membrane 880

(Larsen et al. 2011). This indicates more efficient mitochondrial ATP synthesis since a larger 881

portion of the membrane potential is contributing to ATP synthesis as opposed to uncoupling 882

28

processes. The reduction in membrane leak was attributed to a significant reduction in ANT 883

protein expression and a strong trend for a reduction in UCP-3 protein expression (Larsen et al. 884

2011). However, Whitfield et al. (2016) found dramatically different results where 7 days of BRJ 885

supplementation showed no effect on mitochondrial efficiency as expressed by no change in leak 886

respiration, UCP-3 or ANT protein content, and the P/O ratio. Although it is possible there are 887

different mechanisms of action for NaNO3 compared to BRJ supplementation, this is highly 888

unlikely and more research is required to understand these stark differences, and to elucidate the 889

effects of dietary NO3- on mitochondrial coupling and efficiency. 890

Alternatively, it has been suggested that NO is capable of reversibly inhibiting complex 891

IV of the ETC by competing with O2 at its binding site (Brown and Cooper 1994; Cleeter et al. 892

1994; Bolaños et al. 1994; Guiffrè et al. 2000). By changing the stoichiometry between O2 used 893

and ATP produced, this would result in a decrease in O2 utilization and would ultimately 894

decrease the O2 cost of exercise. 895

Mitochondrial Biogenesis 896

A few cell culture studies have identified NO as a stimulus for mitochondrial biogenesis. 897

These studies have demonstrated an increase in mitochondria DNA, mitochondrial markers such 898

as cytochrome oxidase IV (COXIV) and cytochrome C, and increased O2 consumption following 899

incubation with NO (Nisoli et al. 2003; 2004). It is thought that NO acts through Gc and cGMP 900

to activate peroxisome proliferator-activated receptor gamma coactivator a-alpha (PGC-1𝛼), the 901

master regulator of mitochondrial biogenesis (Nisoli et al. 2004; Lira et al. 2010). However, 902

these findings have not been replicated with BRJ supplementation in humans. Larsen et al. 903

(2011) found no change in mitochondrial density or mRNA content for proteins related to 904

mitochondrial biogenesis (PGC-1𝛼, mitochondrial transcription factor A (TFAM), COXIV) 905

29

following 3 days of NaNO3 supplementation. These results are not surprising as the time course 906

for significant mitochondrial biogenesis to occur is likely longer than this supplementation 907

period. Furthermore, since significant reductions in the O2 cost of exercise occur merely 2.5 h 908

following BRJ supplementation it is likely that the mechanism of action is at least partially 909

independent of increased protein content as this does not occur on this timescale. Moreover, 910

Whitfield et al. (2016) found no change in mitochondrial protein content following 7 days of 911

BRJ supplementation in recreationally active males, suggesting that BRJ supplementation may 912

not influence mitochondrial biogenesis in humans. 913

Improved Contractile Efficiency 914

Reduction in the ATP Cost of Skeletal Muscle Force Production 915

The ATP cost of skeletal muscle force production is dominated by sarcoplasmic 916

reticulum calcium ATPase (SERCA) and myosin ATPase which account for ~25 – 30% and 70% 917

of the total cost of contraction respectively (Barclay et al. 2007). Na+-K+ ATPase is shown to 918

account for ~5% of the ATP cost of skeletal muscle force production (Barclay et al. 2007). 919

Therefore, due to their robust contributions to the ATP cost of exercise, it was a logical step to 920

investigate the effect of dietary NO3- supplementation on the ATP cost associated with SERCA 921

and myosin ATPase. 922

Recently, a number of studies have contributed to the hypothesis that BRJ 923

supplementation may reduce the ATP cost of exercise. However, the exact mechanism of action 924

remains to be elucidated. Bailey et al. (2010) designed an elegant study to indirectly tease apart 925

the potential mechanisms of action associated with the reduced O2 cost of exercise following 926

BRJ supplementation. These authors used magnetic resonance spectroscopy (MRS) to determine 927

the total ATP cost of low and high intensity knee extension exercise, and estimated the relative 928

30

ATP contributions from PCr, glycolysis, and oxidative phosphorylation. These authors found a 929

significant reduction in total ATP hydrolysis during low intensity and high intensity exercise 930

with BRJ supplementation and demonstrated a 25% improvement in exercise tolerance during 931

high intensity exercise to exhaustion following BRJ supplementation. These findings suggest that 932

the O2 cost of submaximal exercise may be reduced through an improvement in skeletal muscle 933

contractile efficiency. Using a similar methodology, Fulford et al. (2013) also found a significant 934

decrease in the relative contribution of PCr to ATP hydrolysis during knee extension exercise. It 935

is important to note that these findings are based on calculations and have inherent error built 936

into them. Furthermore, Fulford et al. (2013) did not calculate the ATP contribution from aerobic 937

or glycolytic metabolism making it difficult to interpret whether BRJ supplementation decreased 938

total ATP turnover or shifted fuel utilization. 939

Other studies have used force frequency curves to assess the effects of BRJ 940

supplementation on skeletal muscle force production and contractile efficiency. Hernandez et al. 941

(2012) found a significant increase in low frequency force production at 10 Hz following 7 days 942

of NaNO3 supplementation in mice. This was accompanied by an increase in intracellular 943

calcium and an increase in the expression of calsequestrin and dihydropyridine receptor proteins, 944

both associated with calcium-handling. Haider and Folland (2014) found an increase in force 945

production at a low stimulation frequency of 10 Hz following 7 days of BRJ supplementation in 946

healthy males. Whitfield et al. (2017) reported similar findings where low stimulation frequency 947

force production was increased at 10 Hz in healthy males suggesting improved contractile 948

efficiency. However, unlike Hernandez et al. (2012), this was not associated with changes in 949

calcium-handling protein expression suggesting BRJ may influence the force produced via 950

myosin ATPase during skeletal muscle contraction. Conversely, Hoon et al. (2015) found no 951

31

effect of 4 days of BRJ supplementation on skeletal muscle force production in 19 healthy adults 952

(13 males). The discrepancies in the outcomes from these studies may be explained by the 953

variation in study model as well as the supplementation and electrical stimulation protocols, and 954

by the small magnitude of the expected changes. 955

Since dietary NO3- supplementation can reduce the oxygen cost of exercise merely 2.5 h 956

following an acute dose, it is unlikely that protein content changes in this time frame. Therefore, 957

it still remains to be elucidated if dietary NO3- supplementation results in post-translational 958

modifications that may alter the activity of calcium-handling or contractile proteins. There is 959

evidence to suggest that dietary NO3- supplementation may slow cross bridge cycling to allow 960

for greater force production per ATP hydrolyzed. Using skinned skeletal muscle fibers from 961

rabbits and differentiated myotubes from mice, Nogueira et al. (2009) demonstrated that NO was 962

able to s-nitrosylate myosin on multiple cysteine thiols. This was accomplished by adding 963

reactive nitrogen species to the reaction mixtures. These authors found that exposure to NO 964

resulted in decreased myosin ATPase activity but no change in the affinity of myosin for actin 965

suggesting a slowing of cross bridge cycling (Nogueira et al. 2009). This was the first evidence 966

that NO may play a role in increasing skeletal muscle force production via altering myosin 967

ATPase function. This work was promptly followed up by Evangelista et al. (2010) who 968

confirmed that exposure of rat skeletal muscle myosin to NO donors can s-nitrosylate cysteine 969

residues on myosin which caused a slowing of cross bridge cycling and ultimately doubled force 970

production. 971

Reactive Oxygen Species (ROS) 972

A study by Whitfield et al. (2016) demonstrated increased intermyofibrillar (IMF) 973

mitochondrial ROS production following 7 days of BRJ supplementation. These authors 974

32

suggested that increased ROS production may influence the skeletal muscle contractile 975

machinery by improving mechanical efficiency and ultimately increasing force production 976

during cross bridge cycling. Alterations in mitochondrial ROS production is an attractive theory 977

for the mechanism underlying BRJ supplementation because it helps explain the acute effects 978

and the diminished effect or lack of effect seen in elite athletic populations. These populations 979

have increased superoxide dismutase (SOD) content and activity which may buffer the 980

mitochondrial ROS production and mitigate any performance benefits of BRJ supplementation. 981

Unfortunately, these authors found no correlation between increased mitochondrial ROS 982

production and the decrease in whole body VO2 during submaximal exercise, suggesting that the 983

in vitro experimental results may not explain the whole-body effects of BRJ supplementation. 984

Clearly future work needs to investigate the in vivo effect of BRJ supplementation and 985

mitochondrial ROS production, and the effects on calcium-handling as well as skeletal muscle 986

contractile machinery. It is possible that ROS produced at the level of the mitochondria may 987

influence the ATP cost of the contractile apparatus. 988

Women – Addressing the Gender Gap 989

Throughout history women have been consistently underrepresented in sport medicine 990

and exercise research, and only recently has this gender gap begun to be attenuated. Costello et 991

al. (2014) indicated that between 2011 and 2013, women still only represented 39% of research 992

participants across 1382 studies. Furthermore, it is widely accepted that sexual dimorphism 993

exists in human physiology and metabolic responses to exercise making it extremely valuable to 994

study sex specific responses to exercise interventions. Unfortunately, a major barrier to including 995

women in exercise studies is the added complexity of the menstrual cycle and the hormonal 996

profiles associated with this process. Hormonal contraceptive use is a method used to minimize 997

33

hormonal fluctuations throughout the menstrual cycle, however this only became common 998

practice in the 1980s (Liao 2012) further contributing to the delay in active recruitment of female 999

research participants. 1000

Characterization of the Menstrual Cycle 1001

A typical female menstrual cycle is ~28 days in duration and is typically characterized by 1002

the early follicular phase (days 1 – 7), late follicular phase (days 7 – 14), early luteal phase (days 1003

14 – 21), and late luteal phase (days 21 – 28). Menses is classified as day 1 of the menstrual 1004

cycle. Estrogen and progesterone levels follow an inverse relationship throughout the menstrual 1005

cycle (Figure 5). During the early follicular and late follicular phase estrogen levels continue to 1006

rise until a peak at ovulation (day 14). Following ovulation estrogen levels decline and 1007

progesterone levels begin to rise until menses occurs again (Jonge et al. 2003). Research utilizing 1008

female participants is further complicated by abnormal menstrual cycle durations, assumption of 1009

normal hormone fluctuations, and the individual variability associated with menstrual cycle 1010

phases (Jonge et al. 2003). The most reliable method to confirm menstrual cycle phase is by 1011

measuring plasma hormone concentrations using commercially available kits (Jonge et al. 2003). 1012

1013

Figure 5: Hormonal profile throughout a normal menstrual cycle with significant surges in 1014 estrogen (estradiol) and progesterone (Sherman and Korenman 1975). 1015 1016 1017

34

NO Varies Throughout the Menstrual Cycle 1018

Some work has investigated the variation in NO production that occurs naturally 1019

throughout the menstrual cycle and there are discrepancies in the literature as to which female 1020

sex hormone, estrogen or progesterone, is the main regulator of NO production. Some authors 1021

suggested that NO metabolites are higher in the follicular phase and peak at ovulation when 1022

estrogen concentrations are at their highest (Cicinelli et al. 1996). Alternatively, it has also been 1023

proposed that NO production is inhibited by progesterone which peaks during the luteal phase 1024

(Giusti et al. 2002; Teran et al. 2002). In line with these hypotheses, Williams et al. (2001) 1025

demonstrated that brachial artery flow mediated dilatation was higher during the follicular phase, 1026

peaking near ovulation and dropping off sharply in the early luteal phase. This suggests a 1027

possible positive correlation between estrogen concentrations and NO production, as well as a 1028

potential inhibitory effect of progesterone on NO production. Lastly, when exhaled NO is 1029

measured there appears to be either no correlation of sex hormones with NO metabolites (Morris 1030

et al. 2002), or a relationship where estrogen decreases NO production and progesterone 1031

increases NO production (Mandhane et al. 2009). It is possible that these discrepancies exist 1032

because exhaled NO is a less reliable measurement of NO metabolites than blood parameters. 1033

Cell culture studies have proposed a role of estrogen in stimulating the production of NO 1034

as there are estrogen receptors located on NOS (Xia and Krukoff 2004; Townsend et al. 2011). 1035

Xia and Krukoff (2004) showed that the addition of estrogen to neuroblastoma cells resulted in 1036

the activation of eNOS and nNOS, and ultimately facilitated the production of NO. Similarly, 1037

Townsend et al. (2011) demonstrated that when human bronchial epithelial cells were exposed to 1038

estrogen there was a rapid increase in NO production, whereas administration of an estrogen 1039

receptor antagonist resulted in inhibition of NO production. These authors proposed that estrogen 1040

35

activates caveolin-1 which is able to increase intracellular Ca2+ concentrations and can ultimately 1041

activate NOS to produce NO (Townsend et al. 2011). It still remains to be elucidated whether 1042

this relationship exists in skeletal muscle. 1043

Pharmacokinetics of Hormonal Contraceptives 1044

Studies have sought to characterize the estrogen and progesterone profiles in women 1045

actively using hormonal contraceptives and it is clear that hormonal contraceptives providing 1046

synthetic estrogen and progesterone prevent ovulation by attenuating the surge in estrogen that 1047

typically occurs around day 14 of a natural menstrual cycle (Gaspard et al. 1983; Kuhl et al. 1048

1985; Jung-Hoffmann et al. 1988; Christin-Maitre et al. 2010). Hormonal contraceptive use 1049

ultimately results in stable and low estrogen levels throughout the menstrual cycle (Christin-1050

Maitre et al. 2010) (Figure 6). 1051

1052

Figure 6: Low estrogen (estradiol) and progesterone levels throughout the menstrual cycle in 1053 women actively using two different forms of hormonal birth control (Christin-Maitre et al. 1054 2010). 1055

36

Previous Female BRJ Literature 1056

There is a major gender gap in BRJ exercise research with only 5 studies that specifically 1057

investigated female populations. This is in stark comparison to the more than 45 studies with 1058

strictly male populations. Of the 5 female studies that exist, there is a diverse range of exercise 1059

modalities, fitness levels, and supplementation protocols. 1060

The first study providing insight that women may respond positively to acute and chronic 1061

dietary NO3- supplementation was performed by Vanhatalo et al. (2010) who demonstrated a 1062

~5% reduction in the O2 cost of moderate intensity exercise in a group of 5 males and 3 females 1063

following 2.5 h, 5 days and 15 days of supplementation. Although these authors do not provide 1064

the individual data points or comment on potential sex differences in the responses to BRJ 1065

supplementation, it is highly likely that most of the female subjects responded positively to this 1066

intervention. 1067

Three studies in strictly female populations have investigated the effects of BRJ 1068

supplementation on exercise performance in trained athletes. However, none of these studies 1069

measured the O2 cost of submaximal exercise. One study explored the effects of acute BRJ 1070

supplementation, sodium phosphate and the combination on repeated sprint performance and 1071

simulated game play in 13 female amateur team-sport athletes (Buck et al. 2015). The subjects 1072

ingested 6 mM dietary NO3- 3 h prior to the exercise protocol. Despite a significant rise in 1073

plasma NO3-, there was no effect of BRJ supplementation on repeated sprint performance. These 1074

authors did not measure plasma NO2- to give an indication whether NO3- was being converted to 1075

NO2- via the enterosalivary circulation. It is possible that these women did not respond to BRJ 1076

supplementation because the dose was too low to elicit a positive effect on exercise performance 1077

or the women may have been too trained. 1078

37

Similarly, Glaister et al. (2015) explored the effects of acute BRJ, caffeine, and the 1079

combination on 20 km cycling TT performance in 14 trained female cyclists. Approximately 2.5 1080

h prior to exercise, the subjects consumed ~7.3 mM dietary NO3-. Despite significant rises in 1081

plasma NO3- and NO2-, there was no effect of BRJ supplementation on TT performance. 1082

Interestingly, these authors found plasma NO2- to increase from 92 to 297 nM, whereas Lansley 1083

et al. (2011) administered an acute dose of 6.2 mM dietary NO3- and found plasma NO2- to 1084

increase from 293 to 575 nM. In contrast to the work of Glaister et al. (2015), Lansley et al. 1085

(2011) demonstrated a significant improvement in 16.1 km cycling TT performance in trained 1086

males. This may suggest that there is a plasma [NO2-] threshold that dietary NO3- 1087

supplementation must surpass in order to elicit a biological effect. 1088

Lastly, Pospieszna et al. (2016) explored the effects of chronic BRJ supplementation on 6 1089

sets of 50 m swimming sprints, and 800 m swimming performance in 11 female collegiate 1090

swimmers. The subjects participated in two, 8 day supplementation periods where they 1091

supplemented with either 0.5 L of BRJ and chokeberry juice or 0.5 L of carrot juice with added 1092

KNO3. Each supplement delivered 10.5 mM of dietary NO3- per day. A major flaw of this study 1093

was that no PLA treatment was utilized for comparison and proper blinding. Nonetheless, these 1094

authors found that both chronic BRJ and chokeberry supplementation as well as carrot juice 1095

supplementation with added KNO3 improved repeated sprint performance by 3.13% and 2.09% 1096

respectively. Specifically, both juices improved performance on sprints 4 through 6. The 1097

improvements were significantly greater with BRJ and chokeberry supplementation compared to 1098

carrot juice with added KNO3 supplementation. Furthermore, both juices significantly reduced 1099

the time to complete the 800 m swim, with greater improvements seen following carrot juice 1100

supplementation with added KNO3. It is possible that dietary NO3- elicited a positive effect on 1101

38

performance in this study since the daily dose was significantly greater than that of Buck et al. 1102

(2015) and Glaister et al. (2015) and the supplementation protocol lasted for 8 days instead of an 1103

acute dose. However, we cannot rule out the possibility of a placebo effect influencing the 1104

outcome of this study. 1105

To date, only two studies have investigated the effects of BRJ supplementation on 1106

exercise economy and performance in recreational or sedentary female populations. First, Bond 1107

Jr et al. (2014) explored the effects of acute BRJ supplementation on submaximal VO2 and blood 1108

pressure in 12 sedentary African American women (VO2peak, 26.1 + 3.3 mL/kg/min). The women 1109

were provided with either ~12 mM dietary NO3- or orange juice (negligible NO3- content) 2 h 1110

prior to blood sampling and exercise testing for 5 min each at 40, 60 and 80% VO2peak. Blood 1111

pressure was measured during exercise. These authors found significant increases in plasma NO 1112

following acute BRJ supplementation. Furthermore, there was a significant reduction in SBP at 1113

rest as well as during exercise at 40, 60 and 80% VO2peak. BRJ supplementation had no effect on 1114

VO2 at rest, but reduced VO2 by ~15% at 40, 60, and 80% VO2peak (as inferred from a figure in 1115

the manuscript). A major limitation of this study is the lack of a NO3--free PLA, and these 1116

authors failed to report the mean VO2 values associated with rest and exercise at 40, 60 and 80% 1117

VO2peak. 1118

Rienks et al. (2015) was the only other group to investigate the effects of acute BRJ 1119

supplementation on VO2 during exercise. The major outcome of this study was to determine the 1120

effects of acute BRJ supplementation on work performed during a rating of perceived exertion 1121

(RPE) clamp protocol in 10 recreationally active females (VO2peak, 36.1 + 4.7 mL/kg/min). To do 1122

this, the subjects ingested 12.9 mM dietary NO3- 2.5 h prior to cycling exercise. These authors 1123

measured VO2 during a 20 min cycling protocol where subjects exercised at a self-selected 1124

39

intensity that elicited an RPE of 13 (somewhat hard). A secondary outcome of this study was to 1125

determine the effect of acute BRJ supplementation on submaximal O2 uptake. This was assessed 1126

by 5 min at 75 W to measure constant workload VO2 following a 5 min cooldown after the 20 1127

min RPE clamp protocol. There was no effect of BRJ supplementation on the work completed or 1128

total VO2 during the 20 min RPE clamp protocol. However, acute BRJ supplementation resulted 1129

in a weak but statistically significant 4% reduction in VO2 while cycling at 75 W (p = 0.048). 1130

Interestingly, only Bond Jr et al. (2014) and Buck et al. (2015) attempted to control for 1131

menstrual cycle phase, and the selected phases differed between these two studies where Bond Jr 1132

et al. (2014) utilized the luteal phase and Buck et al. (2015) utilized the follicular phase. 1133

Furthermore, Pospieszna et al. (2016) was the only group to investigate chronic BRJ 1134

supplementation, and no study to date has combined acute and chronic supplementation into the 1135

same study protocol with strictly female participants. Lastly, only Rienks et al. (2015) recruited 1136

recreationally active subjects when the bulk of the literature suggests the reductions in VO2 are 1137

present in recreationally active males compared to their trained counterparts. Therefore, of the 5 1138

studies, three show improvement following BRJ supplementation. However, the results and 1139

validity of these studies are clouded by poor study design and poorly presented data. 1140

Conclusion 1141

Ultimately, there is very little research exploring the effects of BRJ supplementation in 1142

female populations making it difficult to draw comprehensive conclusions on exercise economy 1143

and performance. It is important to have a number of studies from a variety of laboratories to 1144

validate and support the existing research findings in sedentary, recreationally active as well as 1145

trained female subjects. Furthermore, it is important to explore the effectiveness and mechanisms 1146

behind BRJ supplementation in female populations as these have yet to be examined. 1147

40

Chapter 2: Statement of the Problem 1148

Rationale 1149

Over the last decade, a wealth of research has surfaced exploring the effects of NO3--rich 1150

BRJ on athletic performance across a multitude of exercise modalities. The vast majority of this 1151

research has been conducted on male participants, and it appears that the greatest benefits are 1152

seen in recreationally active males. Moreover, there are only 5 BRJ studies that have considered 1153

exclusively female subjects, and only 2 of those studies measured submaximal O2 uptake. Only 1154

one of these studies recruited recreationally active women, however their primary outcome was 1155

performance during a RPE clamp protocol. Furthermore, none of these studies assessed both 1156

acute and chronic BRJ supplementation in the same protocol. Therefore, the purpose of this 1157

study was to determine the effects of acute and chronic BRJ supplementation on submaximal 1158

cycling economy, and performance on a 4 kJ/kg time trial in recreationally active females. 1159

BRJ was used instead of sodium NO3- or potassium NO3- because BRJ is a commercially 1160

available product that athletes from around the globe, and across a range of calibres, use to 1161

potentially enhance their athletic performance. 1162

The subjects supplemented with BRJ and PLA both acutely and chronically to determine 1163

if short-term and long-term supplementation periods had differing effects on submaximal 1164

exercise economy and time trial performance in recreationally active women. Beneficial effects 1165

have been seen in recreationally active males both acutely and chronically. 1166

The subjects were required to ingest 13 mmol NO3- approximately 2.5 h before the 1167

exercise protocol on the mornings of the experimental trials. This dose has been shown to reduce 1168

the O2 cost of exercise and improve time trial performance in recreationally active males, 1169

whereas doses smaller than 4.2 mmol NO3- have not shown these same benefits (Wylie et al. 1170

41

2013). Secondly, pharmacokinetic profiles have demonstrated that NO3- levels peak in the blood 1171

approximately 2.5 h after consumption of BRJ (Wylie et al. 2013). On the chronic 1172

supplementation days, the subjects were instructed to consume 26 mmol NO3- as 13 mmol in the 1173

morning and 13 mmol in the evening. Following a dose of 13 mmol NO3- plasma NO3- 1174

concentrations have been shown to return to baseline 12 h post-ingestion, therefore the subject’s 1175

plasma NO3- concentrations were constantly elevated during this supplementation regime. This 1176

dose was selected as it is higher than any other female BRJ study to ensure adequate 1177

accumulation in the blood. 1178

The subjects completed a submaximal exercise protocol at wattages that elicited 50% 1179

VO2peak and 70% VO2peak to assess exercise economy at multiple submaximal workloads. 1180

Secondly, the subjects completed a 4 kJ/kg time trial as a measure of athletic performance. A 1181

time trial was used instead of a time to exhaustion protocol because of greater repeatability 1182

across trials and greater ecological validity (Currell and Jeukendrup 2008). The 4 kJ/kg time trial 1183

was selected instead of a fixed time or fixed distance time trial to normalize the subjects’ work to 1184

their body weight. 1185

Overall, the testing protocol was carefully selected to allow for a comprehensive analysis 1186

of the effects of BRJ supplementation on exercise economy and time trial performance in 1187

recreationally active females. 1188

Hypotheses 1189

1) Plasma [NO3-] and [NO2-] will be significantly elevated following acute and chronic 1190

supplementation with NO3--rich BRJ compared to baseline and PLA in recreationally active 1191

females. 1192

42

2) Acute and chronic supplementation with NO3--rich BRJ will significantly decrease the O2 cost 1193

of submaximal cycling at 50% VO2peak and 70% VO2peak in recreationally active females. 1194

3) Acute and chronic supplementation with NO3--rich BRJ will improve performance on a 4 1195

kJ/kg time trial in recreationally active females. 1196

Chapter 3: Methods 1197

Subjects 1198

Twelve recreationally active females were recruited to participate in this study (mean + 1199

SEM, age: 23 + 0.4 years; weight: 65.0 + 3.0 kg; height: 169 + 2 cm; VO2peak: 40.7 + 1.2 1200

mL/kg/min). Subjects were included in the study if they were healthy, recreationally active 1201

females, between the ages of 18 and 30, actively using hormonal contraceptives for 6 months 1202

prior to participation, and consumed less than 300 mg NO3- per day. Subjects were considered 1203

recreationally active if they were physically active 3 – 4 times per week and had a VO2peak of 1204

~ 35 – 45 mL/kg/min. Subjects reported being physical active 4 + 0.2 days per week and 1205

performed 4.5 + 1.0 h of exercise/week in the 6 months leading up to participation in this study. 1206

The subjects were informed of the study requirements and potential risks prior to providing oral 1207

and written consent. This study was approved by the University of Guelph Research Ethics 1208

Board (#REB17-02-008). 1209

Study Design 1210

This study was comprised of 7 laboratory visits (Figure 7). The first visit consisted of a 1211

VO2peak test to determine the subject’s aerobic capacity, and familiarization trials were performed 1212

during visits two and three. Then, using a double-blind, randomized, crossover design, the 1213

subjects supplemented with BRJ (6.5 mmol NO3-/70 mL; Beet It Sport, James White Drinks, 1214

Ipswitch, UK) or a NO3--free placebo (PLA, 0.006 mmol NO3-/70 mL; Beet It Sport) for 1 day 1215

43

(acute) and 8 days (chronic). The treatment conditions were separated by a washout period (9 + 1 1216

days). This time course had been previously validated to allow sufficient time for plasma NO3- 1217

and NO2- levels to return to baseline following chronic supplementation with exogenous NO3- 1218

(Nyakayiru et al. 2017). The PLA was manufactured using an ion resin exchange that selectively 1219

removed NO3- from the original product (Lansley et al. 2011). The BRJ and PLA were 1220

indistinguishable by taste, smell, appearance, and packaging. The supplementation order was 1221

counterbalanced, where 6 subjects began supplementing with BRJ and 6 began supplementing 1222

with PLA. To investigate the effects of acute and chronic BRJ supplementation on exercise 1223

performance, the subjects completed a submaximal cycling protocol and an aerobic-based time 1224

trial (TT) on days 1 and 8 of supplementation. The testing began with subjects consuming 140 1225

mL of BRJ (13 mmol NO3-) or PLA 2.5 h prior to submaximal cycling and the TT. In the 1226

evening on day 1, the subjects consumed an additional 140 mL of BRJ (13 mmol NO3-) or PLA. 1227

On days 2 – 7, the subjects consumed 280 mL of BRJ or PLA. The first dose of 140 mL was 1228

consumed in the morning with breakfast and the second 140 mL dose was consumed in the 1229

evening with dinner. 1230

1231

Figure 7: A schematic of the experimental design consisting of 7 laboratory visits. Subjects 1232 completed 2 x 8 days supplementation phases separated by a washout of 9 + 1 days. The subjects 1233 came to the laboratory on days 1 (acute trial) and 8 (chronic trial) of each supplementation phase 1234 to perform a cycling protocol consisting of submaximal cycling and an aerobic-based time trial. 1235 1236 Pre-Experimental Tests 1237

To determine VO2peak, and the cycling intensity to elicit 50% and 70% VO2peak, each 1238

subject performed an incremental exercise test to volitional exhaustion on a cycle ergometer 1239

44

(Lode Excalibur Sport, Groningen, The Netherlands). The subjects were equipped with a heart 1240

rate (HR) monitor (POLAR H10, Oulu, Finland) before warming up for 2 min at 60 W, then the 1241

power output (PO) was increased to 100 W. Once the subjects achieved steady state VO2 (~ 2 1242

min) at each stage, the PO was increased by 25 W. Steady state was defined as two x 20 s VO2 1243

measurements that were within 50 mL O2/min of each other. This process was repeated until the 1244

subjects reached volitional exhaustion (VO2peak was determined as the highest value obtained 1245

during the test). Time, HR, VO2, and PO were recorded at the end of each step increment. VO2 1246

and carbon dioxide production (VCO2) were measured (Moxus Modular VO2 System, 1247

Pittsburgh, USA) in 20 s intervals during the test. 1248

The subjects completed two familiarization trials prior to the experimental trials. The 1249

subjects arrived to the laboratory and were asked to provide a urine sample for determination of 1250

urine specific gravity (USG). Then, the subjects were weighed and equipped with a HR monitor. 1251

In the first familiarization trial the subjects were set up comfortably on the ergometer and the 1252

position was documented for subsequent trials. During the familiarization trials, the subjects 1253

completed the entire experimental exercise protocol consisting of a 5 min warm up at 60 W, 10 1254

min at 50% VO2peak, 10 min at 70% VO2peak, and a 4 kJ/kg body mass (BM) aerobic time-based 1255

TT. In the first familiarization trial the subjects consumed water ad libitum. The subjects were 1256

weighed after the trial for determination of body mass loss through sweat. If the subject lost or 1257

gained mass compared to the initial weigh in, then the volume of fluid provided during the 1258

exercise protocol was adjusted prior to the second familiarization trial. The volume of water 1259

consumed in the second familiarization trial was replicated for the subsequent experimental 1260

trials. The subjects moved to the experimental trials once there was less than 5% variability 1261

45

between the TT performances (Currell and Jeukendrup 2008). All subjects’ TT performances 1262

achieved this, therefore no subject was required to complete a third familiarization trial. 1263

1264

Diet, Sleep and Exercise Standardization 1265

The subjects recorded their dietary intake, sleep and exercise in the 48 h leading up to the 1266

familiarization and experimental trials. Subjects were asked to replicate their diet, sleep and 1267

exercise habits for the duration of the study. In the 24 h prior to the familiarization and 1268

experimental trials, the subjects were asked to refrain from intense exercise and alcohol, to 1269

abstain from caffeine in the 12 h before the trials. Subjects were not instructed to abstain from 1270

NO3- rich foods, as the pre-study screening indicated that they did not consume diets high in 1271

dietary NO3-. The use of antibacterial mouthwash and chewing gum was prohibited during the 1272

experimental trials, as there is evidence suggesting that these substances destroy the anaerobic 1273

bacteria in the mouth necessary for the conversion of NO3- to NO2- and NO (McDonagh et al. 1274

2015). Furthermore, the subjects were instructed to avoid brushing their teeth for 2 h before and 1275

after the ingestion of the BRJ or PLA. 1276

For the experimental trials, the subjects arrived to the laboratory in an overnight fasted 1277

state. A baseline blood sample (9.5 mL) was drawn for analysis of serum estrogen and 1278

progesterone as well as plasma [NO3-] and [NO2-]. The subjects were then provided with a 1279

standardized breakfast (consumed in < 15 min) that consisted of a banana, a SoLo GIâ bar, and 1280

140 mL of BRJ or PLA. This breakfast was intentionally carbohydrate-rich to ensure that 1281

carbohydrate was not limiting during exercise (79 g CHO, 16.5 g protein, 7.7 g fat). Following 1282

the standardized breakfast, the subjects were required to wait 2 h before a second blood sample 1283

was drawn (6 mL) for determination of plasma [NO3-] and [NO2-]. During this period, the 1284

46

subjects were provided with water to ensure adequate hydration prior to exercise. The volume of 1285

water consumed at rest in the first experimental trial was replicated for subsequent trials. 1286

1287

Experimental Exercise Protocol 1288

Immediately after the second blood sample, the subjects were escorted to the exercise 1289

laboratory (21.4 + 0.3oC, 56.0 + 3.0%). The subjects were asked to provide a urine sample for 1290

determination of USG and to void their bladder. They were then weighed and equipped with a 1291

HR monitor. Exercise testing occurred ~ 2.5 h post-beverage ingestion (Figure 8). The exercise 1292

protocol was designed to make VO2 measurements at two different submaximal exercise 1293

intensities, followed by an aerobic TT. The subjects completed a 5 min warmup at 60 W, 1294

followed by 10 min at 50% VO2peak (81 + 4 W) and 10 min at 70% VO2peak (131 + 5 W). The 1295

subjects were instructed to maintain a cadence of 80 revolutions per minute (rpm) throughout the 1296

submaximal cycling protocol. The subjects then rested for 3 min before completing a 4 kJ/kg BM 1297

TT (260 + 12 kJ). The ergometer was set to linear mode, and the subjects started cycling at a 1298

resistance equal to 70% VO2peak at 80 rpm. The subjects were able to increase their PO by 1299

increasing their rpms and were only shown the number of kJ completed during the TT. 1300

The rating of perceived exertion (RPE) was recorded every 5 min throughout the 1301

submaximal exercise protocol using the Borg Scale (6 – 20) (Borg 1982). Respiratory 1302

measurements were recorded over 20 s intervals for the final 4 min of each submaximal exercise 1303

intensity. Time, PO, rpm and RPE were recorded with the completion of each 20% of the TT. 1304

Verbal encouragement was provided with each 20% completed. HR was recorded continuously 1305

throughout the exercise protocol. Following the time trial, subjects completed a blinding and 1306

adverse side effects questionnaire. 1307

47

1308

1309

1310 Figure 8: Experimental trial exercise protocol. Red arrows indicate recording of rating of 1311 perceived exertion (RPE), power output (PO) and revolutions per minute (rpm) at 5 min intervals 1312 during the submaximal exercise protocol. Blue arrows indicate collection of oxygen uptake for 1313 the final 4 min of each submaximal exercise intensity. Black arrows indicate recording of RPE, 1314 PO, rpm and time elapsed with every 20% of the 4 kJ/kg body mass (BM) time trial completed. 1315 1316 Blood Sampling and Analysis 1317

On the mornings of the experimental trials, the subjects arrived to the laboratory and had 1318

a baseline blood sample drawn from the antecubital vein. This blood sample was ~9.5 mL, of 1319

which 3.5 mL was collected into a serum separator tube and 6 mL was collected into a K2EDTA-1320

coated tube. The second 6 mL blood sample was collected 2 h later into a K2EDTA-coated tube. 1321

Immediately after collection, all blood tubes were thoroughly mixed. 1322

Immediately following collection, the K2EDTA-coated tubes were centrifuged for 10 min 1323

at 2600 rpm and 4oC. The plasma was then aliquoted equally into two 1.8 mL cryovials. The 1324

serum separator tube was left for 30 min at room temperature to coagulate and centrifuged for 10 1325

min at 3000 rpm and 4oC. The serum was aliquoted equally into two 1.8 mL cryovials. All 1326

plasma and serum samples were immediately stored at -80oC for future analysis. 1327

Serum samples were analyzed for estradiol (pg/mL) and progesterone (ng/mL) 1328

concentrations using commercially available ELISA kits (Abcam, Item Nos: ab108667, 1329

ab108654, USA). For determination of serum estradiol concentrations, the serum samples were 1330

thawed on ice and vortexed for 5 s before 25 µL of sample or standard was added to the 96 well 1331

plate and 200 µL of Estradiol-horseradish peroxidase (HRP) conjugate was added to each well. 1332

48

The plate was covered and incubated for 2 hr at 37oC to allow the Estradiol-HRP to compete 1333

with the estradiol within the sample for antibody binding sites. The wells were then aspirated and 1334

washed three times with 300 µL of 1x wash solution to remove unbound material and 100 µL 1335

substrate was added to each well. The substrate was catalyzed by 3,3’,5,5’-Tetramethylbenzidine 1336

(TMB) to produce blue colouration. The plate was incubated for 30 min at room temperature 1337

before 100 µL stop solution was added to each well. The absorbance was read at 450 nm. 1338

For determination of serum progesterone concentrations, the serum samples were thawed 1339

on ice and vortexed for 5 s before 20 µL of sample or standard was added to the 96 well plate. 1340

Next, 200 µL of Progesterone-HRP conjugate was added to each well. The plate was covered 1341

and incubated for 1 hr at 37oC to allow the Progesterone-HRP to compete with the progesterone 1342

within the sample for antibody binding sites. The wells were then aspirated and washed three 1343

times with 300 µL of 1x washing solution to remove unbound material and 100 µL substrate was 1344

added to each well. The substrate was catalyzed by TMB substrate to produce blue colouration. 1345

The plate was incubated at room temperature for 15 min before 100 µL stop solution was added 1346

to each well. The absorbance was read at 450 nm. 1347

Plasma samples were analyzed for NO3- and NO2- concentrations using 1348

chemiluminescence. To quantify NO2-, the plasma sample was passed with a gastight syringe 1349

into a reduction solution containing 45 mmol/L potassium iodide and 10 mmol/L iodine in 1350

glacial acetic acid. The reduction solution was kept at 56oC and continuously bubbled with 1351

nitrogen. Sample aliquots were incubated with sulfanilamide for 15 min. Sulfanilamide forms a 1352

stable diazonium ion that traps NO2- and prevents it from further reduction to NO. The quantity 1353

of NO2- was determined by calculating the area under the curve for NO2- from the aliquots 1354

treated with sulfanilamide and subtracting this from the NO2- area under the curve for the 1355

49

untreated aliquots. Plasma NO3- was determined by reducing NO3- to NO via vanadium (III) 1356

chloride. The amount of NO3- was quantified by subtracting the previously determined NO2- 1357

concentration from the amount of NO3- reduced to NO (Lundberg and Govoni 2004). 1358

Compliance 1359

Each subject’s adherence to the chronic supplementation protocol was assessed through 1360

the return of the empty supplemental beverage containers, and through the analysis of plasma 1361

NO3- and NO2- concentrations. Subjects were instructed to return all empty supplement 1362

containers at the time of their chronic supplementation trial. 1363

Statistical Analysis 1364

Diet records were analyzed using Food Processor Nutrition Analysis Software (ESHA 1365

Research, Salem, USA). Statistical analyses were performed using Prism 7 (GraphPad Software, 1366

San Diego, USA). Statistical significance was achieved when P < 0.05. Data are presented as 1367

mean + SEM. The statistical tests for serum estradiol and progesterone, pre-trial characteristics 1368

(48 h diet and exercise record, pre-trial sleep), environmental conditions (temperature, humidity), 1369

hydration measures (pre-fluid intake, USG, during-fluid intake), submaximal exercise respiratory 1370

variables (VO2, VCO2, RER, VE, VT, and f), energy expenditure, and submaximal exercise 1371

performance (HR, RPE, and rpm) were conducted using a two-way analysis of variance 1372

(ANOVA) and Tukey’s multiple comparisons post-hoc test. 1373

Time trial learning effect was assessed with a one-way ANOVA and Tukey’s multiple 1374

comparisons post-hoc test. Time trial performance (HR, RPE, PO, rpm, and elapsed time) were 1375

analyzed using a three-way ANOVA (BRJ vs. PLA, acute vs. chronic, time) and Tukey’s 1376

multiple comparisons post hoc test. 1377

50

Responders to BRJ supplementation were arbitrarily defined as individuals who 1378

demonstrated a ³ 3% reduction in VO2 as this adheres with previous research and is greater than 1379

the coefficient of variation (CV) between the second familiarization trial, Acute PLA condition, 1380

and Chronic PLA condition. Responders to BRJ supplementation were also arbitrarily defined as 1381

individuals who demonstrated a ≥ 4.2% improvement in time trial performance as this is greater 1382

than the CV between the second familiarization trial, Acute PLA condition, and Chronic PLA 1383

condition. 1384

Chapter 4: Results 1385

Blood Parameters 1386

Estradiol and Progesterone 1387

Serum estradiol and progesterone concentrations were reported for 10 subjects. The data 1388

for 2 subjects was lost during analysis due to values below the limit of detection. Serum estradiol 1389

concentrations were not significantly different across the trials (Acute PLA, 0.01 + 0.003: 1390

Chronic PLA, 0.02 + 0.01: Acute BRJ, 0.01 + 0.01: Chronic BRJ, 0.01 + 0.01 pg/mL). Serum 1391

progesterone concentrations were not significantly different across the trials (Acute PLA, 0.98 + 1392

0.36: Chronic PLA, 0.47 + 0.13: Acute BRJ, 1.39 + 0.97: Chronic BRJ, 1.14 + 0.76 ng/mL). 1393

Nitrate (NO3-) 1394

Baseline plasma NO3- concentrations were not different in the Acute PLA (36 + 4 µM), 1395

Chronic PLA (36 + 3 µM), and Acute BRJ trials (44 + 3 µM) (Figure 9). However, plasma NO3- 1396

was significantly elevated at baseline in the Chronic BRJ trial (548 + 57 µM) compared to all 1397

other conditions (p < 0.0001). There was no significant difference in plasma NO3- 2 h post-1398

beverage ingestion in the Acute PLA (49 + 5 µM) and Chronic PLA (40 + 3 µM) trials. 1399

Conversely, there was a significant rise in plasma NO3- concentrations 2 h post-beverage 1400

51

ingestion in the Acute BRJ (776 + 33 µM) and Chronic BRJ (1125 + 65 µM) trials (p < 0.0001). 1401

Plasma NO3- concentrations were significantly greater in the Acute BRJ trial compared to Acute 1402

PLA and Chronic PLA 2 h post-beverage consumption (p < 0.0001). Similarly, 2 h post-1403

beverage consumption, plasma NO3- concentrations were significantly greater in the Chronic 1404

BRJ trial compared to Acute PLA and Chronic PLA (p < 0.0001). Interestingly, plasma NO3- 1405

concentrations were significantly greater in the Chronic BRJ compared to the Acute BRJ trial 2 h 1406

post-beverage consumption (p < 0.0001). 1407

1408

Figure 9: Plasma nitrate (NO3-) at baseline (B) and two hours post-ingestion (2 h) of 140 mL 1409 beetroot juice (BRJ) or NO3--free placebo (PLA) both acutely and chronically. Values are mean 1410 + SEM. *, significantly different than all other conditions. 1411 1412 Nitrite (NO2-) 1413

Plasma NO2- concentrations were not significantly different at baseline across all trials 1414

(Acute PLA, 231 + 28: Chronic PLA, 216 + 27: Acute BRJ, 240 + 26: Chronic BRJ, 328 + 20 1415

Acute

PLA (B)

Acute

PLA (2 h)

Chronic

PLA (B)

Chronic

PLA (2 h)

Acute

BRJ (B)

Acute

BRJ (2 h

)

Chronic

BRJ (B)

Chronic

BRJ (2 h

)0

50

100

500

800

1100

1400

NO

3- (µM

) *

*

*

52

nm) (Figure 10). There was no change in plasma NO2- concentrations 2 h post-beverage 1416

ingestion in the Acute PLA (266 + 23 nm) and Chronic PLA (280 + 24 nm) trials. The increase 1417

in plasma NO2- 2 h post-beverage ingestion was significant in both the Acute BRJ (729 + 80 nm) 1418

and Chronic BRJ (706 + 95 nm) trials (p < 0.0001). However, there was no significant difference 1419

in plasma NO2- between the Acute BRJ and Chronic BRJ trials 2 h post-beverage ingestion. 1420

1421

Figure 10: Plasma nitrite (NO2-) at baseline (B) and two hours post-ingestion (2 h) of 140 mL 1422 beetroot juice (BRJ) or nitrate-free placebo (PLA) both acutely and chronically. Values are mean 1423 + SEM. †, significantly greater than conditions with no symbol. 1424 1425 Pre-Trial Characteristics 1426

The subjects successfully maintained their dietary habits throughout the study as there 1427

was no significant difference in energy intake in the 48 h prior to the experimental trials (Acute 1428

PLA, 2447 + 210: Chronic PLA, 2150 + 104: Acute BRJ, 2155 + 190: Chronic BRJ, 2316 + 179 1429

kcal). Similarly, there was no change in physical activity in the 48 h prior to each trial. Lastly, 1430

Acute

PLA (B)

Acute

PLA (2 h)

Chronic

PLA (B)

Chronic

PLA (2 h)

Acute

BRJ (B)

Acute

BRJ (2 h

)

Chronic

BRJ (B)

Chronic

BRJ (2 h

)0

200

400

600

800

1000

NO

2- (nM

)

† †

53

there was no difference in sleep patterns the night prior to each trial (Acute PLA, 7.6 + 0.2: 1431

Chronic PLA, 7.3 + 0.2: Acute BRJ, 7.4 + 0.2: Chronic BRJ, 7.2 + 0.3 h). 1432

Environmental Characteristics 1433

The average temperature in the exercise laboratory during the exercise tests was 21.4 + 1434

0.3oC and the average relative humidity was 56.0 + 3.0%. Temperature did not differ between 1435

any of the trials (Acute PLA, 21.4 + 0.3: Chronic PLA, 21.5 + 0.4: Acute BRJ, 21.3 + 0.2: 1436

Chronic BRJ, 21.6 + 0.3oC), and there was no significant difference in relative humidity across 1437

the trials (Acute PLA, 55.3 + 2.9: Chronic PLA, 53.8 + 3.4: Acute BRJ, 58.6 + 2.8: Chronic 1438

BRJ, 56.2 + 3.1%). 1439

Hydration Status 1440

On average, the subjects consumed 665 + 49 mL of water in the 2 h prior to the exercise 1441

trial. The volume of water consumed prior to exercise did not differ between the trials (Acute 1442

PLA, 677 + 43: Chronic PLA, 674 + 47: Acute BRJ, 658 + 51: Chronic BRJ, 651 + 58 mL). The 1443

subjects arrived to the exercise laboratory very well hydrated with an average USG of 1.005 + 1444

0.0012. USG was not different across trials (Acute PLA, 1.003 + 0.001: Chronic PLA, 1.005 + 1445

0.002: Acute BRJ, 1.005 + 0.001: Chronic BRJ, 1.005 + 0.001). On average, the subjects 1446

consumed 569 + 56 mL of water throughout the exercise protocol. The volume of water 1447

consumed during the exercise protocol was unchanged across trials (Acute PLA, 576 + 56: 1448

Chronic PLA, 558 + 57: Acute BRJ, 584 + 62: Chronic BRJ, 560 + 54 mL). The subjects were 1449

successful at replenishing their sweat loss with water, as the average change in body mass was 1450

+ 0.1 + 0.1%. The percent change in BM was the same across trials (Acute PLA, + 0.1 + 0.1: 1451

Chronic PLA, + 0.2 + 0.1: Acute BRJ, + 0.2 + 0.1: Chronic BRJ, + 0.1 + 0.1%). 1452

Exercise Economy 50% VO2peak 1453

54

There was no change in mean VO2 between the trials when the subjects cycled at an 1454

intensity that elicited 50% VO2peak (Acute PLA, 1254 + 45: Chronic PLA, 1267 + 45: Acute 1455

BRJ, 1259 + 46: Chronic BRJ, 1252 + 42 mL/min O2) (Figure 11). Interestingly, 25% of the 1456

subjects were defined as responders to Acute BRJ supplementation and 42% of the subjects were 1457

defined as responders to Chronic BRJ supplementation at 50% VO2peak (Figure 12). A responder 1458

was defined as a subject who demonstrated a ≥ 3% reduction in VO2 following BRJ 1459

supplementation compared to PLA. This is in accordance with previous research (Wylie et al. 1460

2013) and outside of the calculated CV for VO2 at 50% VO2peak (2.4%). In addition, VE, VT, f, 1461

and RER were unchanged across trials (Table 1). Furthermore, there was a main effect of 1462

condition where there was a small but significant increase in VCO2 in the chronic trials (Chronic 1463

PLA, 1160 + 35: Chronic BRJ, 1172 + 36 mL/min) compared to the acute trials (Acute PLA, 1464

1147 + 33: Acute BRJ, 1155 + 43 mL/min) (p = 0.01) (Table 1). HR was not significantly 1465

different between conditions or from 5 - 10 min at 50% VO2peak (Table 2). RPE was not 1466

significantly different between conditions, however there was a main effect of time where RPE 1467

was significantly increased from 5 - 10 min at 50% VO2peak (p < 0.0001) (Table 2). The subjects 1468

successfully maintained a cadence of 80 rpm at 50% VO2peak during each of the trials (Table 2). 1469

There was no change in calculated energy expenditure between trials (Acute PLA, 62 + 2: 1470

Chronic PLA, 63 + 2: Acute BRJ, 62 + 2: Chronic BRJ, 62 + 2 kcal). 1471

1472

55

1473

Figure 11: Mean oxygen uptake (VO2) at 50% peak oxygen uptake (VO2peak) following acute and 1474 chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Data reported 1475 as mean + SEM. 1476 1477

1478 Figure 12: Individual oxygen uptake (VO2) at 50% peak oxygen uptake (VO2peak) following 1479 acute and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). 1480 1481 Exercise Economy 70% VO2peak 1482

Mean VO2 remained unchanged at 70% VO2peak between trials (Acute PLA, 1804 + 60: 1483

Chronic PLA, 1814 + 54: Acute BRJ, 1791 + 53: Chronic BRJ, 1789 + 54 mL/min O2) (Figure 1484

13). Interestingly, 25% of the subjects were defined as responders to Acute BRJ supplementation 1485

Acute

Chronic

1000

1200

1400

1600

1800

VO2 (

mL/

min

)

PLA

BRJ

56

and 33% of the subjects were defined as responders to Chronic BRJ supplementation (Figure 1486

14). A responder was defined as a subject who demonstrated a ≥ 3% reduction in VO2 following 1487

BRJ supplementation compared to PLA. This is in accordance with previous research (Wylie et 1488

al. 2013) and outside of the calculated CV for VO2 at 70% VO2peak (2.0%). Regardless of trial, 1489

VCO2, VT, f, and RER were unchanged (Table 1). However, there was a very small but 1490

significant decrease in VE in the BRJ trials (Acute BRJ, 42.4 + 1.5: Chronic BRJ, 42.6 L/min) 1491

compared to PLA (Acute PLA, 43.4 + 1.6: Chronic PLA, 44.4 + 1.4 L/min) (p = 0.002). HR was 1492

not significantly different between conditions or from 5 - 10 min at 70% VO2peak (Table 2). RPE 1493

was not significantly different between conditions, however there was a main effect of time 1494

where RPE increased from 5 - 10 min at 70% VO2peak (p < 0.0001) (Table 2). The subjects 1495

successfully maintained a cadence of 80 rpm at 70% VO2peak (Table 2). Energy expenditure was 1496

unaltered between trials (Acute PLA, 90 + 3: Chronic PLA, 90 + 3: Acute BRJ, 89 + 3: Chronic 1497

BRJ, 89 + 3 kcal). 1498

1499

Figure 13: Mean oxygen uptake (VO2) at 70% peak oxygen uptake (VO2peak) following acute and 1500 chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Data reported 1501 as mean + SEM. 1502

57

1503 Figure 14: Individual oxygen uptake (VO2) at 70% peak oxygen uptake (VO2peak) following acute 1504 and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). 1505 1506 Time Trial Performance 1507

There was no learning effect associated with time trial completion (Trial 1, 1857 + 107: 1508

Trial 2, 1873 + 112: Trial 3, 1913 + 117: Trial 4, 1912 + 120 s) (Figure 15). There was no 1509

significant difference in time trial completion across the trials (Acute PLA, 1845 + 71: Chronic 1510

PLA, 1869 + 75: Acute BRJ, 1912 + 93: Chronic BRJ, 1892 + 98 s) (Figure 16). Similarly, there 1511

was no difference in the time to complete each 20% split between trials (Figure 17). There was a 1512

main effect of time in which the final split was significantly faster than the other splits (p = 1513

0.0001). No difference was observed for HR throughout the time trial (Figure 18). However, a 1514

main effect of time was demonstrated, where HR was significantly higher in the final split 1515

compared to the rest of the time trial (p < 0.0001). There was no effect of condition on RPE, but 1516

RPE was significantly higher in the final split compared to the rest of the time trial (p < 0.0001) 1517

(Figure 19). There was no significant difference in rpm or PO between conditions during the 1518

time trial (Figure 20). However, there was a main effect of time in which rpm (p < 0.0001) and 1519

PO (p < 0.0001) were significantly increased in the final split compared to the rest of the time 1520

trial. 1521

Acute

Chronic

1400

1600

1800

2000

2200

2400VO

2 (m

L/m

in)

PLA

BRJ

58

1522

Figure 15: Time trial learning effect. Data reported as mean + SEM. 1523 1524

1525 Figure 16: Mean time trial completion. Values reported as mean + SEM. 1526 1527

59

1528 Figure 17: Mean time to complete each 20% split of the time trial. Values reported as mean + 1529 SEM. *, significantly faster than the previous splits. 1530 1531

1532 1533 Figure 18: Mean heart rate (HR) at the end of each 20% split during the time trial. Values 1534 reported as mean + SEM. *, significantly higher than previous splits. 1535 1536

20%

40%

60%

80%

100%

0

100200300

350

400

450Ti

me

(s)

Acute PLA

Acute BRJ

Chronic PLA

Chronic BRJ*

20%

40%

60%

80%

100%

150

160

170

180

190

200

HR

(bpm

)

Acute PLA

Acute BRJ

Chronic PLA

Chronic BRJ

*

60

1537 Figure 19: Mean rating of perceived exertion (RPE) at the end of each 20% split during the time 1538 trial. Values reported as mean + SEM. a, significantly greater than 20%; b, significantly greater 1539 than 20% and 40%; c, significantly greater than every other split. 1540 1541

1542 Figure 20: Mean power output at the end of each 20% split during the time trial. Values reported 1543 as mean + SEM. *, significantly higher than previous splits. 1544 1545 Compliance, Blinding and Adverse Events 1546

The subjects were 100% compliant to the supplementation protocol as assessed through 1547

the return of empty supplemental beverage containers, and plasma NO3- and NO2- analysis. The 1548

results from the blinding and adverse events questionnaire indicated that 100% of the subjects 1549

were successfully blinded to the treatments they received, and 58% of the subjects experienced 1550

mild adverse effects acutely and chronically with both treatments. Nausea was the most 1551

20%

40%

60%

80%

100%

12

14

16

18

20R

PEAcute PLA

Acute BRJ

Chronic PLA

Chronic BRJ

c

ba

a

20%

40%

60%

80%

100%

100

150

200

250

Pow

er O

utpu

t (W

)

Acute PLA

Acute BRJ

Chronic PLA

Chronic BRJ

*

61

commonly reported side effect (41%), 17% reported gastrointestinal upset, 17% reported 1552

beeturia, and 8% reported acid reflux. 1553

Table 1: Respiratory variables at 50% and 70% VO2peak following acute and chronic 1554 supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values reported as 1555 mean + SEM. *, Chronic trials demonstrated significantly higher carbon dioxide production 1556 (VCO2) compared to acute trials. **, BRJ significantly decreased ventilation (VE) compared to 1557 PLA. VT, tidal volume. 1558

50% VO2peak 70% VO2peak

VO2 (mL/min)

Acute PLA 1254 + 45 1804 + 60

Chronic PLA 1267 + 45 1814 + 54

Acute BRJ 1259 + 46 1791 + 53

Chronic BRJ 1252 + 42 1789 + 54

VCO2 (mL/min)

Acute PLA 1147 + 33 * 1697 + 46

Chronic PLA 1160 + 35 1709 + 46

Acute BRJ 1155 + 43 * 1706 + 53

Chronic BRJ 1172 + 36 1709 + 46

RER

Acute PLA 0.917 + 0.008 0.945 + 0.009

Chronic PLA 0.917 + 0.007 0.943 + 0.008

Acute BRJ 0.919 + 0.009 0.953 + 0.006

Chronic BRJ 0.937 + 0.007 0.956 + 0.008

VE (L/min)

Acute PLA 28.2 + 1.0 43.4 + 1.6

Chronic PLA 29.0 + 0.9 44.4 + 1.4

Acute BRJ 27.8 + 1.1 42.6 + 1.4 **

Chronic BRJ 28.1 + 1.0 42.4 + 1.5 **

VT (mL)

Acute PLA 1331 + 71 1588 + 79

Chronic PLA 1325 + 66 1613 + 69

Acute BRJ 1326 + 58 1581 + 63

Chronic BRJ 1319 + 55 1574 + 59

Frequency (#/min)

Acute PLA 27 + 2 34 + 2

Chronic PLA 27 + 1 34 + 2

Acute BRJ 26 + 1 33 + 2

Chronic BRJ 27 + 1 33 + 2

62

Table 2: Heart rate (HR), rating of perceived exertion (RPE) and revolutions per minute (rpm) 1559 recorded at 5 and 10 min at 50% and 70% peak oxygen consumption (VO2peak) following acute 1560 and chronic supplementation with beetroot juice (BRJ) or a nitrate-free placebo (PLA). Values 1561 reported as mean + SEM. 1562 1563

50% VO2peak 70% VO2peak 5 min 10 min 5 min 10 min

HR (bpm)

Acute PLA 132 + 14 133 + 15 160 + 15 164 + 15

Chronic PLA 133 + 10 131 + 10 160 + 10 165 + 11

Acute BRJ 133 + 11 133 + 13 160 + 13 164 + 14

Chronic BRJ 134 + 11 132 + 12 160 + 13 163 + 14

RPE

Acute PLA 9.6 + 2.0 10.1 + 1.8 12.6 + 0.8 13.5 + 0.8

Chronic PLA 9.8 + 1.9 10.7 + 0.5 12.6 + 0.9 13.8 + 0.9

Acute BRJ 10.2 + 1.7 10.2 + 1.7 12.4 + 1.4 13.5 + 1.2

Chronic BRJ 9.3 + 2.2 10.3 + 2.3 12.3 + 1.5 13.5 + 1.3

rpm

Acute PLA 82 + 0.4 81 + 0.3 80 + 0.3 80 + 0.3

Chronic PLA 81 + 0.3 81 + 0.3 81 + 0.3 80 + 0.1

Acute BRJ 81 + 0.5 80 + 0.2 81 + 0.3 81 + 0.3

Chronic BRJ 81 + 1.0 81 + 0.3 80 + 0.3 80 + 0.3

1564 1565

1566

1567

1568

1569

1570

1571

1572

1573

1574

63

Chapter 5: Discussion 1575

The principle findings of this study were that acute and chronic dietary NO3- 1576

supplementation, in the form BRJ, did not improve submaximal exercise economy or aerobic TT 1577

performance in recreationally active females. This study is novel as it is the first to investigate 1578

the effects of acute and chronic BRJ supplementation in females and is the first to address the O2 1579

cost of submaximal exercise in recreationally active females at multiple intensities. 1580

Comparison to Previous Work in Women 1581

There has been little previous BRJ literature in female populations and the results have 1582

produced equivocal findings. The first study to provide insight that women may respond 1583

positively to BRJ supplementation was performed by Vanhatalo et al. (2010). The study cohort 1584

consisted of 5 healthy males and 3 healthy females (VO2peak ~ 47 mL O2/kg/min), and these 1585

authors found a 5% reduction in the O2 cost of moderate intensity submaximal exercise on days 1586

1, 5 and 15 of supplementation with 5.2 mM dietary NO3-. Interestingly, this group demonstrated 1587

significantly elevated plasma NO2- levels prior to supplementation (~454 nM) compared to the 1588

current study (~250 nM). These baseline differences may be attributed to dietary habits or fitness 1589

status. Additionally, the subjects had a smaller rise in plasma NO2- following supplementation 1590

(~650 nM) compared to the current study (~725 nM). The differences in the rise in plasma NO2- 1591

likely reflect the differences in the BRJ doses between the studies. The dramatically different 1592

VO2 outcomes between this study and the current study may reflect the inclusion of 1593

predominantly male subjects in the study by Vanhatalo et al. (2010). 1594

To date, three studies have investigated the effects of BRJ supplementation in trained 1595

female populations. Glaister et al. (2015) found no effect of acute BRJ supplementation on 20 1596

km cycling TT performance (~35 min) in trained female cyclists. Similar to the results from the 1597

64

current study, we found no effect of BRJ supplementation on TT performance lasting ~30 min. 1598

However, it is also important to consider that Glaister et al. (2015) utilized a significantly smaller 1599

dose of BRJ (5.1 mM) which may explain the lack of effect in their study. Similar to Glaister et 1600

al. (2015), Buck et al. (2015) found no effect of acute BRJ supplementation on a 1 h exercise 1601

circuit involving repeated sprints in trained female team sport athletes. Again, it is possible that 1602

the BRJ dose was too small (6 mM) to elicit an ergogenic effect. Both Glaister et al. (2015) and 1603

Buck et al. (2015) obtained plasma samples for determination of NO3- and NO2- levels. 1604

Unsurprisingly, plasma NO3- and NO2- levels were significantly lower following 1605

supplementation compared to the current study. Lastly, Pospieszna et al. (2016) found a 1606

significant improvement in repeated sprint swimming performance as well as 800 m swimming 1607

performance in college level female swimmers with 8 days of BRJ supplementation. These 1608

authors utilized a larger dose of BRJ (10.2 mM) and this is the only study to employ a chronic 1609

supplementation protocol in female subjects. However, it is difficult to determine the validity of 1610

this study as the authors did not use a placebo supplement and no plasma samples were collected. 1611

Bond Jr et al. (2014) measured cycling exercise economy in sedentary, overweight, 1612

African American women. These authors found a significant reduction in the O2 cost of exercise 1613

at 40, 60, and 80% VO2peak following acute BRJ supplementation (~12 mM). This dose is 1614

comparable to the acute 13 mM dose administered in the current study. This may further 1615

emphasize the importance of administering a large enough dose of dietary NO3- in order to 1616

ensure a biological effect is elicited. However, these authors failed to report the VO2 data adding 1617

question to the validity of their results. Rienks et al. (2015) found no effect of acute BRJ 1618

supplementation (12.9 mM) on exercise performance during a 20 min RPE clamp protocol in 1619

recreationally active women. However, as a secondary outcome, these authors measured exercise 1620

65

economy at 75 W for 5 min immediately following the RPE clamp protocol and found a weak, 1621

but statistically significant ~4% reduction in the O2 cost of exercise (p = 0.048). The findings 1622

from these two studies are in contrast to the results from the current study, but inherent study 1623

design flaws and weak statistical significance make it difficult to be confident in the findings 1624

from these studies. Future work from across a number of laboratories is required to determine the 1625

true biological effect of dietary NO3- supplementation in female populations. 1626

Interestingly, the findings from the current study are in stark contrast to the consistent 1627

reports of a reduced O2 cost of exercise in recreationally active men following both acute and 1628

chronic BRJ supplementation (Bailey et al. 2009; Vanhatalo et al. 2010; Lansley et al. 2011; 1629

Wylie et al. 2013). Although the exact mechanism still remains to be elucidated, it is proposed 1630

that dietary NO3- supplementation improves exercise economy and TT performance by 1631

improving contractile, calcium-handling, or mitochondrial efficiency through the provision of 1632

NO (Bailey et al. 2010; Larsen et al. 2011; Hernandez et al. 2012; Whitfield et al. 2016). The 1633

results from this study suggest that there may be differences in the way women utilize dietary 1634

NO3- compared to men. It is possible that NO3- supplementation fails to elicit a response in 1635

recreationally active women because there may be differences in the way dietary NO3- is 1636

digested and handled or differences in the efficiency of NO production. Alternatively, sex 1637

differences in skeletal muscle composition, and the role of estrogen on mitochondria and 1638

contractile machinery may play an important role in determining the effectiveness of NO3- 1639

supplementation. 1640

Nitrate Digestion 1641

It is unlikely that recreationally active women have impaired function of the NO3--NO2--1642

NO pathway. A couple of insights can be drawn by comparing the data from this study to another 1643

66

study from our laboratory that utilized the same absolute dose of BRJ in recreationally active 1644

males as well as the same NO3- and NO2- analytic techniques (Whitfield et al. 2016). In this 1645

study, the relative dose of dietary NO3- was 0.40 mM/kg, whereas Whitfield et al. 2016 1646

implemented a relative dose of 0.33 mM/kg dietary NO3-. In the current study, baseline plasma 1647

NO3- and NO2- levels were 44 uM and 240 nM respectively and Whitfield et al. (2016) found 1648

baseline plasma NO3- and NO2- to be ~25 uM and ~200 nM respectively. Perhaps unsurprisingly, 1649

following acute BRJ supplementation we found plasma NO3- and NO2- levels to increase to 776 1650

uM and 729 nM respectively, whereas Whitfield et al. (2016) reported plasma NO3- and NO2- 1651

levels to increase to ~500 uM and ~500 nM respectively. This supports the notion that the greater 1652

relative dose of dietary NO3- in the current study resulted in a greater increase in plasma NO3- 1653

and NO2- and suggests that women do not have an impaired ability to convert dietary NO3- to 1654

NO2-. This is an interesting finding as 58% of the participants in this study reported mild adverse 1655

side effects associated with BRJ supplementation, with 42% of participants specifically reporting 1656

gastrointestinal (GI) upset. Beets are considered a high Fermentable Oligosaccharides 1657

Disaccharides Monosaccharides and Polyols (FODMAP) food due to their oligosaccharide 1658

content (Marcason 2012). High FODMAP foods are known to cause GI upset due to their poor 1659

digestibility (Magge and Lembo 2012). Women are more likely to be diagnosed with GI 1660

conditions such as irritable bowel syndrome (Anbardan et al. 2012; Oshima and Miwa 2015) 1661

therefore it was hypothesized that women may have more adverse responses to high FODMAP 1662

foods and may not be reaping the benefits of BRJ supplementation due to poor digestibility. 1663

However, it is important to consider that this may be confounded by the fact that young women 1664

are more likely to report GI discomfort and are more likely to consult a physician than young 1665

men (Wang et al. 2013) and based on the dramatic increases in plasma NO3- and NO2- following 1666

67

acute and chronic BRJ supplementation in this study it is unlikely that women have an impaired 1667

ability to process dietary NO3-. 1668

Nitrate and Nitrite Levels 1669

When comparing the baseline plasma NO3- and NO2- levels in this study to the findings 1670

of Whitfield et al. (2016) there are no discernable differences between the sexes. This is in 1671

contrast to findings by Kapil et al. (2010) who noted that although men and women did not differ 1672

in baseline plasma NO3-, women had significantly higher plasma NO2- levels compared to men. 1673

Independent of plasma NO3- and NO2- increases, we cannot rule out the possibility that 1674

women may rely more heavily on endogenous NO production via NOS or that women may have 1675

different dietary habits than men. First, it is possible that women rely more heavily on 1676

endogenous NO production since previous literature suggests a positive link between estrogen 1677

and endogenous NO production (Cicinelli et al. 1996). This would mean a greater reliance on 1678

endogenous NO production and decreased reliance on exogenous provision of dietary NO3-. 1679

Second, it’s possible that women have different dietary habits than men. A study by Jonvik et al. 1680

(2017) demonstrated that absolute dietary NO3- intake was higher in female athletes compared to 1681

male athletes, and when dietary NO3- intake was expressed relative to total energy intake these 1682

differences were more pronounced. However, it is unlikely that the women in this study had 1683

different dietary habits than the men in the study by Whitfield et al. (2016) as the baseline 1684

plasma NO3- and NO2- levels were similar. Furthermore, our exclusion criteria stated that 1685

participants could not consume a high dietary NO3- diet (> 300 mg dietary NO3-/day). It is 1686

unclear if Whitfield et al. (2016) controlled for dietary NO3- habits. 1687

1688

1689

68

Antioxidant Capacity 1690

It has been suggested that estrogen plays an important role in enhancing antioxidant 1691

capacity in women by regulating the activity of antioxidant enzymes. A number of studies have 1692

demonstrated that women have lower plasma levels of markers of oxidative stress than men (Ide 1693

et al. 2002; Bloomer and Fisher-Wellman 2008). Furthermore, a study by Barp et al. (2002) 1694

demonstrated that female rats have lower levels of oxidative stress in heart tissue than male rats, 1695

and this was associated with female rats have greater enzyme activity of superoxide dismutase 1696

(SOD) and glutathione peroxidase (GPx). Interestingly, it is likely that estrogen drives this 1697

relationship as it has been shown that women who undergo hysterectomies (Barp et al. 2002; 1698

Borras et al. 2003; Bellanti et al. 2013) as well as post-menopausal women (Singh et al. 2016) 1699

have reduced SOD enzyme content (Borras et al. 2003; Bellanti et al. 2013; Singh et al. 2016) 1700

and activity (Barp et al. 2002; Borras et al. 2003). 1701

It is possible that the increased antioxidant capacity seen in young, healthy women 1702

buffers the reactive nitrogen species and subsequent s-nitrosylation associated with BRJ 1703

supplementation. However, it is important to consider that the antioxidant capacity relationships 1704

described above were observed in plasma blood samples as well as heart, liver and brain tissue 1705

and may not reflect skeletal muscle. If these sex differences in antioxidant capacity exist in 1706

skeletal muscle, women may have minimized effects of BRJ supplementation on altering skeletal 1707

muscle contractile function, calcium-handling, or mitochondrial efficiency. Future work is 1708

needed to elucidate whether these differences exist at the level of the skeletal muscle and if they 1709

manifest in significant metabolic and physiological outcomes. 1710

1711

1712

69

Differences in Skeletal Muscle Fiber Types and Capillarization 1713

A number of studies have performed histochemical analysis on human skeletal muscle 1714

fibers to determine sex differences in fiber type composition. These studies consistently 1715

demonstrate that women have an ~5% greater proportion of type I oxidative muscle fibers, 1716

whereas men have a greater proportion of type II glycolytic muscle fibers (Staron et al. 2000; 1717

Haizlip et al. 2014). BRJ supplementation is purported to elicit its beneficial effects 1718

predominantly in type II muscle fibers. This is attributed to the fact that the NO3--NO2--NO 1719

pathway is an O2 independent pathway that elicits its effects in scenarios of reduced O2 delivery 1720

(Vanhatalo et al. 2011). BRJ has been suggested to facilitate the delivery of O2 to type II muscle 1721

fibers that are less oxygenated than type I muscle fibers (Wilkerson et al. 2012). Therefore, it is 1722

possible that women are less likely to respond to BRJ supplementation due to a greater 1723

proportion of type I muscle fibers, a more oxygenated skeletal muscle environment, and 1724

therefore a decreased reliance on the O2 independent NO3--NO2--NO pathway. Although this is 1725

an interesting hypothesis, it is unclear if a 5% difference in fiber type composition is a large 1726

enough difference to drive these differences in response to BRJ supplementation. 1727

However, this hypothesis is supported in two different ways. First, as previously 1728

described, there is a positive link between estrogen and NOS activity (Cicinelli et al. 1996; 1729

Townsend et al. 2011) suggesting that women may place a heavier reliance on the endogenous 1730

NO production than men, and that the exogenous NO3--NO2--NO pathway may provide a less 1731

robust contribution to NO production in women than men. Secondly, women have greater 1732

skeletal muscle capillarization (Roepstorff et al. 2006; Hoeg et al. 2009) suggesting they have 1733

enhanced O2 delivery to skeletal muscle compared to men. This would further support the notion 1734

of a decreased reliance on the NO3--NO2--NO pathway in women compared to men due to a more 1735

70

oxygenated muscle environment. Conversely, it is important to consider the fact that men have 1736

greater hemoglobin levels (Murphy 2014) and skeletal muscle mass (Janssen et al. 2000), and 1737

these are larger driving factors for O2 delivery and exercise performance than skeletal muscle 1738

capillarization and fiber type differences. Furthermore, it is unlikely that O2 delivery is hindered 1739

at 50% or 70% VO2peak making this hypothesis feasible but unlikely to explain the potential sex 1740

differences seen in this study with BRJ supplementation. 1741

Mitochondrial Efficiency 1742

There is scientific literature suggesting that women have a greater reliance on fat for fuel 1743

during submaximal exercise (Blatchford et al. 1985; Horton 1998; Tarnopolsky et al. 1990). It is 1744

possible that since women rely more heavily on oxidative metabolism, have a greater capillary 1745

density and have a greater proportion of type I muscle fibers that they may exhibit more efficient 1746

mitochondrial coupling compared to men. This notion is in part supported by evidence 1747

suggesting that the livers and brains from female rats produce less mitochondrial H2O2 compared 1748

to males indicating the potential for improved mitochondrial coupling (Borras et al. 2003). 1749

Mitochondrial H2O2 production is increased in situations of elevated electron slippage (Wong et 1750

al. 2017). Therefore, it is possible that a lower level of mitochondrial H2O2 in female rats is 1751

indicative of decreased electron slippage and ultimately improved mitochondrial coupling. 1752

Interestingly, this relationship is likely driven by estrogen as Borras et al. (2003) found increased 1753

mitochondrial H2O2 production in ovariectomized rats and that estrogen replacement therapy 1754

reduced mitochondrial H2O2 production to basal levels. Furthermore, a recent study by Acaz-1755

Fonseca et al. (2017) showed that cerebral cortex mitochondria of male mice have greater UCP-2 1756

expression compared to female mice. It is possible that this is a metabolic adaptation to deal with 1757

greater mitochondrial ROS in male mice compared to females. Although these studies did not 1758

71

investigate these relationships in skeletal muscle, it would be interesting to see if these same 1759

trends exist in this tissue. Based off of these speculations, it is possible that women have a 1760

decreased reliance on the NO3--NO2--NO pathway due to more efficient mitochondrial coupling 1761

and ultimately oxidative metabolism. Therefore, it’s possible that any effects of dietary NO3- 1762

supplementation that may affect mitochondrial efficiency may be lost in healthy women who 1763

may exhibit more efficient mitochondrial coupling compared to men. Although mitochondrial 1764

H2O2 production and UCP protein content are not the only indicators of mitochondrial coupling, 1765

they may be interesting avenues for future research to consider in skeletal muscle. 1766

A recent study by Miotto et al. (2018) investigated sex differences in mitochondrial 1767

respiratory kinetics at rest in human skeletal muscle. These authors noted that women have 1768

decreased ADP sensitivity, as well as greater sensitivity for malonyl CoA inhibition of palmitoyl 1769

CoA supported respiration compared to men. The functional role of these findings remains to be 1770

determined, but this is the first piece of evidence supporting sex differences in mitochondrial 1771

function in human skeletal muscle and therefore may support the notion of studying sex 1772

differences in mitochondrial efficiency with respect to BRJ supplementation. 1773

Calcium-Handling Proteins 1774

A final difference that may explain why women do not respond to BRJ supplementation 1775

is the possible differences that may exist in skeletal muscle calcium-handling. Intriguingly, the 1776

time to peak tension and rate of relaxation are faster in the muscle of human males compared to 1777

females (Wüst et al. 2008). Time to peak tension is used as an indirect measure of calcium 1778

release at the onset of skeletal muscle contraction and rate of relaxation is used as a proxy for the 1779

reuptake of calcium following skeletal muscle contraction. This suggests that men may have a 1780

72

greater ability to release and resequester calcium than women and may implicate sex differences 1781

in skeletal muscle calcium-handling protein content or activity. 1782

A study by Welle et al. (2008) demonstrated that healthy women have a 3.6-fold greater 1783

gene expression of RyR3 in skeletal muscle than healthy men. Although mRNA does not always 1784

translate to protein content, it is possible that women have a greater ability to release calcium via 1785

RyR in skeletal muscle. This seems counterintuitive as it would be expected that if women had 1786

greater RyR content they would have greater Ca2+ release compared to men and likely faster time 1787

to peak tension but, this theory does not account for differences in RyR protein activity or the 1788

responsiveness of female contractile elements to Ca2+ influx. However, if more Ca2+ is released 1789

via RyR with no differences in SERCA or CASQ content or activity, it may take longer to 1790

resequester this Ca2+ helping corroborate slower relaxation times seen in women. This would 1791

also result in a greater ATP cost from SERCA to pump the additional Ca2+. Perhaps most 1792

importantly, it is important to readdress the role of fiber type differences between men and 1793

women. Women have a greater proportion of type I fibers, which are known to have slower time 1794

to peak tension and half relaxation times than type II fibers (Buchthal and Schmalbruch 1970). 1795

Although these fiber type differences are around 5% they likely account for the differences in 1796

contractile properties to a greater extent than potential sex differences in skeletal muscle 1797

contractile protein content or activity. 1798

Individual Responders 1799

Although mean VO2 and time trial performance were unaltered by acute and chronic BRJ 1800

supplementation, individual responders were identified at 50% VO2peak, 70% VO2peak and for TT 1801

performance. Interestingly, 25% of the subjects were defined as responders to Acute BRJ 1802

supplementation and 42% of the subjects were defined as responders to Chronic BRJ 1803

73

supplementation at 50% VO2peak. A responder was defined as a subject who demonstrated a ≥ 3% 1804

reduction in VO2 following BRJ supplementation compared to PLA. This is in accordance with 1805

previous research (Wylie et al. 2013) and is outside of the calculated CV for VO2 at 50% VO2peak 1806

(2.4%). Furthermore, 25% of the subjects were defined as responders to Acute BRJ 1807

supplementation and 33% of the subjects were defined as responders to Chronic BRJ 1808

supplementation at 70% VO2peak. Cumulatively, 2 participants responded to BRJ 1809

supplementation in 3 of the 4 conditions, and 1 participant responded in all BRJ conditions. A 1810

TT responder was defined as a subject who demonstrated a ≥ 4.2% reduction in time to complete 1811

the 4 kJ/kg TT as this was the CV for this measurement. None of the participants demonstrated 1812

this level of improvement, however one subject who demonstrated a VO2 response both acutely 1813

and chronically to BRJ supplementation also demonstrated a 3.3% improvement in TT 1814

performance acutely and a 3.8% improvement chronically. 1815

These results are in agreement with studies by Christensen et al. (2013) and Boorsma et 1816

al. (2014) who identified individual responders to BRJ supplementation in males. In the current 1817

study, responders could not be identified based on baseline or rises in plasma NO3- and NO2- 1818

levels. This is in accordance with the findings by Boorsma et al. (2014). It is unclear why the 1819

individuals in this study responded positively to BRJ supplementation since they had similar 1820

dietary habits, exercise regimes, and fitness statuses compared to the other participants. Future 1821

research is required to elucidate the mechanisms behind individualized responses to BRJ 1822

supplementation. Nonetheless, these findings are important because despite no change in mean 1823

VO2, it is still possible for some women to respond positively to acute and chronic BRJ 1824

supplementation, and it is possible for the improvement in submaximal exercise economy to 1825

translate to slight aerobic TT performance benefits in some individuals. 1826

74

Limitations 1827

The metabolic cart used to collect the data in this study has up to 3% error in VO2 1828

measurement, therefore it is possible that the current methodology was not sensitive enough to 1829

detect small reductions in VO2 following BRJ supplementation. Typically, the reduction in VO2 1830

following BRJ supplementation is in the range of 3 – 5%. In the current study, the CV for VO2 1831

was 2.4% at 50% VO2peak and 2.0% at 70% VO2peak. Therefore, it is possible that some of the 1832

change in VO2 associated with BRJ supplementation may have been masked by inherent error 1833

associated with the metabolic cart. 1834

The present study used a 4 kJ/kg TT (~30 min) to assess exercise performance. This style 1835

of TT has been validated to have greater ecological validity compared to time to exhaustion 1836

protocols (Currell and Jeukendrup 2008). However, this may not be a valid measure of 1837

performance in recreationally active individuals who do not train for TT performance. We 1838

ensured each participant was adequately familiarized with the TT protocol twice and that there 1839

was less than 5% difference in TT completion before they graduated to the experimental trials. 1840

Interestingly, the CV for TT performance between the second familiarization trial, Acute PLA 1841

and Chronic PLA trials, where there presumably there was no effect of condition, was 4.24%, 1842

which is in agreement with Currell and Jeukendrup (2008) suggesting < 5% variability in TT 1843

performance for trained individuals. This indicates that this protocol had high repeatability 1844

despite the recreational status of the participants. 1845

Future Directions 1846

Following the completion of this study, a number of questions remain unanswered 1847

regarding sex differences that may exist with BRJ supplementation. Future work should include 1848

a study similar to that of Porcelli et al. (2015) to verify the findings from the current study and to 1849

75

determine if the effect of training status on the response to BRJ supplementation exists in 1850

women. Second, future work should directly compare the responses to acute and chronic BRJ 1851

supplementation in recreationally active male and female populations. If consistent effects are 1852

seen with VO2 and exercise performance, then muscle biopsies should be obtained to delve into 1853

the mechanisms underpinning the potential sex differences that may exist. Lastly, future work 1854

should investigate the effects of BRJ supplementation in sedentary male and female populations 1855

and should determine if improved fitness status via aerobic exercise training can remove the 1856

potential beneficial effect of BRJ supplementation. Specifically, VO2 should be measured at rest, 1857

50% and 70% VO2peak at baseline, following 4 weeks of training, and again following 10 weeks 1858

of aerobic training. Clearly, incorporating women into BRJ research is in its infancy and there 1859

are a number of avenues yet to be explored, it will be fascinating to see how the field grows over 1860

the next few years and what studies stem from the work presented in this thesis. 1861

Conclusion 1862

The main findings from the current study were that acute and chronic supplementation 1863

with NO3--rich BRJ did not improve submaximal cycling exercise economy or aerobic TT 1864

performance in recreationally active women. These findings are novel because it is the first study 1865

to investigate the effects of acute and chronic BRJ supplementation in women and is the first 1866

study to measure exercise economy at multiple exercise intensities in recreationally active 1867

women. Although we did not perform any mechanistic measures in this study, we can speculate 1868

that there may be sex differences associated with BRJ supplementation. Future research should 1869

continue to investigate the potential sex differences that may exist with dietary NO3- 1870

supplementation and should seek to determine the mechanisms underpinning these differences. 1871

1872

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