k. sletten - performance enhancing effects of nitrate related supplements

Department of Public Health – Sport Science

Aarhus University

November 2013

The ergogenic effects of Nitric Oxide-related

supplementation on human aerobic performance

By

Kristian Sletten – 20083846 ______________________________________________

Submitted: November 2013

Supervisor: Kristian Overgaard

Kristian Sletten 20083846 Aarhus University, extended essay, 2013

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Résumé

Formålet med dette review er systematisk og kvalitativt, at vurdere den foreliggende litteratur vedrørende

nitrid oxid (NO)-relateret supplementering i forhold til human aerob præstation.

En litteratursøgning i to databaser (PubMed, Embase) samt krydsreferering af de aktuelle artikler blev

udført den 19. august 2013, hvor i alt 550 artikler blev identificeret. Efter inklusion og eksklusions

processen var endeligt 23 studier inkluderet. Inklusionskriterierne krævede at studierne skulle indeholde et

NO-relateret substrat i form af rødbede, rødbedesaft, arginine, citrulline eller kunstige NO-relateret

substrater in vivo til raske individer med en aldersvariation på mellem 15-45 år. Studierne skulle ligeledes

have en kontrol eller placebogruppe og skulle være udført under normale omstændigheder. De inkluderede

studier blev kvalitetsvurderet i forhold til en modificeret PEDro-scala og scorede mellem 3 og 7 points ud af

9 mulige hvor hovedparten scorede i omegnen af 6 points.

De inkluderede studier foreslår, at NO-relateret supplementering kan være præstationsfremmende. Det ses

hyppigere ved studier der evaluerer tid til udmattelse testprotokoller og arbejdsøkonomi i form af en

mindre iltoptagelse ved et givent arbejde. Indikationerne for en ergogen effekt syntes at være mere

konsistente ved utrænede fremfor trænede individer og effekten observeres både ved akut og

længerevarende supplementering. Endelig ses det at højintens arbejde giver større ergogene udslag end

ved lavintens arbejde.

Konkluderende påpeger litteraturen evidens for at NO-relateret supplementering kan bidrage ergogent til

aerob humane præstationer. Effekten syntes dog at være afhængig af træningsstatus, typen af arbejde

samt supplementerings strategien.


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Abstract

The purpose of this extended paper was to systematically and qualitatively review the literature relating to

studies on the effects of nitric-oxide (NO)-related supplementation on the aerobic performance of healthy

human individuals.

A literature search of two databases (PubMed, Embase) and cross-referencing of the articles was

conducted on 19 August 2013, resulting in a total of 550 papers being identified. After applying the

inclusion and exclusion criteria 23 studies met the conditions set. These studies were confined to NO-

related supplementation in the form of beetroot, beetroot juice, arginine and citrulline, or a

pharmacological NO substance applied in vivo to healthy individuals in the age range 15-45 years. The

studies likewise included a control or a placebo group and were conducted in normoxic conditions. The

studies included were rated according to a modified PEDro-scale for quality assessment and scored

between 3 and 7 of a maximum 9 points, with the majority of studies scoring around 6 points.

The literature studied proposes that NO-related supplementation can improve exercise performance. This

is especially evident in measurements of time to exhaustion and as increased efficiency by lowering

submaximal oxygen consumption. Indications of an effect are more consistent for untrained than for

trained subjects and an ergogenic effect is obtainable both after acute and prolonged supplementation.

Finally, it seems from data on high intensity exercise that it induces larger effects than low intensity

protocols.

In conclusion, the evidence indicates that NO-related supplements are likely to result in ergogenic effects

on aerobic human performance. However, the general effect seems to depend on training status, exercise

type and supplementation strategy.


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Contents

1. Introduction ............................................................................................................................................... 6

1.1. Purpose and problem ............................................................................................................................. 6

2. History and formation of NO ..................................................................................................................... 7

2.1. History .................................................................................................................................................... 7

2.2. Formation ............................................................................................................................................... 7

2.2.1. NOS-dependent pathway .............................................................................................................. 7

2.2.2. NOS-independent pathway ........................................................................................................... 8

3. Effects and physiological mechanisms of NO ............................................................................................ 8

3.1. NO-related vasodilation ......................................................................................................................... 9

3.2. NO-related 02 utilization ......................................................................................................................... 9

4. Methodology ........................................................................................................................................... 10

4.1. Systematic literature search ................................................................................................................. 10

4.2. Selection criteria and selection of studies ........................................................................................... 11

4.3. PEDro scale ........................................................................................................................................... 12

4.4. Data Extraction and presentation ........................................................................................................ 13

5. Results ...................................................................................................................................................... 14

5.1. General study characteristics ............................................................................................................... 14

5.2. Study Quality ........................................................................................................................................ 17

5.2.1. Summary ...................................................................................................................................... 19

5.3. Training status ...................................................................................................................................... 19

5.3.1. Summary ...................................................................................................................................... 20

5.4. Exercise measurements, duration and intensity .................................................................................. 20

5.4.1. Direct performance measurements ............................................................................................ 20

5.4.2. Indirect performance measurements.......................................................................................... 21

5.4.3. Duration and intensity ................................................................................................................. 22

5.4.4. Summary ...................................................................................................................................... 22

5.5. Supplement, timing and quantity ......................................................................................................... 26

5.5.1. Summary ...................................................................................................................................... 27


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5.6. Overall results summary....................................................................................................................... 27

6. Discussion ................................................................................................................................................ 27

6.1. Training status ...................................................................................................................................... 27

6.1.1. NOS activity in trained subjects ................................................................................................... 28

6.1.2. Development of Hypoxia ............................................................................................................. 29

6.1.3. Summary ...................................................................................................................................... 29

6.2. Exercise measurements, duration and intensity .................................................................................. 30

6.2.1. Direct performance measurements ............................................................................................ 30

6.2.2. Indirect performance measurements.......................................................................................... 30

6.2.3. Duration and intensity ................................................................................................................. 32

6.2.4. Summary ...................................................................................................................................... 33

6.3. Supplement, timing and quantity ......................................................................................................... 33

6.3.1. Supplement ................................................................................................................................. 33

6.3.2. Timing and quantity ..................................................................................................................... 34

6.3.3. Summary ...................................................................................................................................... 36

6.4. Limitations ............................................................................................................................................ 37

7. Conclusion ................................................................................................................................................ 37

8. Recommendations for future areas of research ..................................................................................... 38

9. References ............................................................................................................................................... 39

Total words: /36000

Keystrokes: Max 80 sider 192000


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1. Introduction

1.1. Purpose and problem

The purpose of this extended paper was to systematically and qualitatively review the literature relating to

studies on the effect of nitric-oxide (NO)-related supplementation on aerobic performance in healthy

humans.

Recently, interest has grown in the possible ergogenic effects of NO on human performance due to the fact

that NO can be manipulated easily through diet. In particular, achievable ergogenic amounts of NO have

been supplemented in the form of beetroot juice or beetroot concentrate, which are considered major

natural sources of the precursor nitrate (NO3).

Studies have shown that this supplementation has positive effects on exercise performance-related

measurements (1-14). This improvement has been connected specifically to improved exercise economy

observed in measures of lower oxygen consumption at a given workload (VO2submax). However, the

ergogenic effect has been questioned since some studies have not observed any significant effect when

supplementing with NO-related substances (15-21), and others have even observed an anti-ergogenic

effect (22, 23). Recently, studies employing advances in supplementation strategies and test protocols have

been published, providing even more valid data within this field of research. Even though it still remains to

be defined which factors are the determining variables to benefit from a NO-related supplementation. It is

first and foremost speculated that the ergogenic effects could be dependent on individual training status,

the exercise performed and supplementation strategy. However, only a few studies discuss these

speculations.

On the basis of the controversy and new studies being published with still inconclusive results it seems

relevant to systematically review the literature from a qualitative perspective concerning the ergogenic

effects of nitrate and other NO-related supplements in healthy individuals. This review therefore tries to

sum up and extend the present knowledge by including recently published studies and through deeper

analysis of possible determining variables.


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2. History and formation of NO

2.1. History

Although NO is a naturally occurring substance in the human body, involved in many physiological

processes, it is only in the last two decades that NO has been studied intensively and has been recognized

as a very important cellular signalling molecule (24). Until 1994 NO and its precursor, NO3, were considered

damaging to health and restrictions where made worldwide to limit their ingestion. However, later studies

proposed opposite effects. The earliest health

studies to identify benefits were conducted

by Brown & Cooper (25) who discovered

hitherto unknown functions, which proposed

NO could act as an anti-cytotoxic agent. This

study led to a positive view of NO, and today

it is used in therapy for several human

diseases, including myocardial infarction,

stroke, systemic and pulmonary hypertension,

and gastric ulceration (26). The increasing use

and interest have yielded further knowledge

about the physiological formation and

mechanisms of NO and related substances.

2.2. Formation

As shown in Figure 1 NO is synthesized endogenously in the human body through at least two main

pathways (27): a direct conversion to NO (NOS-dependent), or a reduction from NO3 to nitrite (NO2), to

finally form NO (NOS-independent).

2.2.1. NOS-dependent pathway

The NOS-dependent pathway uses a nitric oxide synthase enzyme (NOS) that catalyzes a complex

enzymatic reaction, leading to the formation of NO, which is dependent on a number of co-factors (28). The

NOS-dependent pathway uses two main substances, the amino acids arginine and citrulline, to increase NO

levels. Arginine is proposed to be the main precursor of the two for NO synthesis due to the fact that

citrulline is a precursor for arginine (27).

Figure 1 - NOS dependent and independent pathways of NO formation


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2.2.2. NOS-independent pathway

As shown in Figure 1 the NOS-independent pathway represents an alternative and simple biochemical

process by reducing NO3 and NO2 to NO, which acts parallel to the classical arginine-NOS-NO pathway. This

reduction is well documented, and several studies show that NO3 or NO3 containing supplements, typically

beetroot or beetroot juice, increase the endogen NO level (4-7). This pathway to increase NO availability

has been described as being especially important in conditions of low oxygen availability, such as during

exercise, where it seems that NO synthesis by the NOS-dependent pathway is impaired (29).

3. Effects and physiological mechanisms of NO

Brown & Cooper’s (25) advance created further interest in NO studies, leading to a positive perspective,

where NO-related supplementation in recent decades has been common in therapeutic situations (26),

particularly to observe increased blood flow (5) and blood pressure drops (30) after ingestion. In particular,

observing increased blood flow in working muscles after NO-related supplementation was interesting for

exercise physiologists who speculated that this could improve aerobic performance by delivering more

blood and thus more oxygen to working muscles. One of the first studies to combine NO supplementation

with exercise was conducted by Larsen et al. (2) who, in 2007, demonstrated that supplementation

improved exercise performance in terms of better exercise efficiency. Their results were particularly

interesting due to the fact that traditional exercise physiology normally dictates that there is minimal

change in oxygen consumption for an individual at a given workload regardless of training status, age or

diet (2). Furthermore, the effects of NO-related supplementation seem even more appealing because the

precursor for NO, that is NO3, exists in a variety of foods, especially in beetroot, which can easily affect NO

levels in our daily diet (31). These recent studies have given rise to a considerable increase in the

consumption of NO3-rich food, in particular beetroot juice, by athletes to obtain ergogenic benefit from an

increased level of NO in the cells.

NO interacts with several possible parameters that could be responsible for an ergogenic effect (32). Some

studies (33, 34) have proposed one or a combination of several effects, which can be summarized as the

following two mechanisms of NO:

1. NO-related vasodilation

2. NO-related O2 utilization


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3.1. NO-related vasodilation

According to Umbrello et al. (33) the proposed ergogenic effect of NO in relation to vasodilation in physical

exercise occurs due to the exercise-induced hypoxic situation in which cells respond to the reduced arterial

oxygen concentration by redistributing oxygen to working tissues. In this process NO is thought to be a

major signalling and effector molecule improving blood flow and thereby oxygen supply by activating cyclic

guanosine monophosphate (cGMP) which eventually leads to smooth muscle relaxation. NO therefore acts

as an endothelium-derived relaxing factor which has under rest conditions been identified in animal (35)

and human studies (36). From this perspective it is hypothesised that NO contribution during exercise helps

the body to deliver oxygen to support ongoing adenosine triphosphate (ATP) synthesis and, in turn, a

constant work output (6).

3.2. NO-related 02 utilization

A reduction of O2 consumption at a given workload has been confirmed in several of the included studies

(2, 3, 7, 8, 13). Bailey et al. (7) offer three possible explanations for reduced O2 cost at a given workload:

1. A possible NO-related mechanism is muscle efficiency improvements in terms of alterations in energy

expenditure in the form of less ATP per muscle contraction. This explanation is based on a number of

studies that present an NO regulatory effect in several interactions in muscle contraction cycle kinetics,

as shown by Galler et al. (37); reduced ryanodine receptor activity and Ca2+ release as described by

Stamler & Meissner (32); and inhibited Ca2+ATPase activity as proposed by Viner et al. (38) . Bailey et al.

(7) suggested that these NO interactions could eventually reduce the O2 cost of exercise, thereby

improving exercise efficiency through reducing the total ATP cost of muscle force production.

2. In mitochondrial respiration where ATP is being resynthesized, Lundberg et al. (26) found several

possible interactions between NO3, NO2, NO and the mitochondrion which could reduce the O2 cost of

exercise. However, the most common explanations for an NO-related ergogenic effect are thought to

involve a reduction in the mitochondria’s pumps and proton slippage where it is believed that an

increase in NO availability decreases the proton uncoupling and down-regulating of the transporters,

uncoupling protein 3 (UCP3) and adenine nucleotide transporter (ANT). These mechanisms eventually

lead to an improved oxygen/power ratio by reducing the O2 cost at a given intensity due to a

mitochondrial efficiency enhancer.


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Table 1 - Detailed list of articles retrieved and the search terms applied in the two databases

3. The last theory, proposed by Bailey et al. (7), states that a lower O2 rate could possibly occur if NO3

were to inhibit mitochondrial ATP production and thereby induce a shift towards non-oxidative

pathways for cellular ATP synthesis. In this case a compensatory increase in energy provision through

substrate-level phosphorylation would be required and result in lower O2 utilization.

However, it seems unlikely that Bailey et al.’s (7) last hypothesis would be the main cause of lower O2 due

to the fact that a steady state of O2 is seen in long trials, and energy use would be the same in the end.

Taking the three explanations into account, Wietzberg et al. (39) argue that lower O2 seems primarily

dependent on the first two explanations. However, the specific mechanism and relations of explanations

for NO’s O2 lowering effect remain to be elucidated.

4. Methodology

4.1. Systematic literature search

This review is based on a systematic literature search of two different databases (PubMed, Embase) that

was performed by one researcher to identify articles about ergogenic effects of NO-related

supplementation published before 19 August 2013. The search was performed using MeSH or Emtree

terms where possible or using a regular text search when no terms were identified. The searches included

the following terms: Exercise, Citrulline, Arginine, Nitrate, Nitric Oxide, Beetroot, Ergogenic, Dietary

supplement. The searches were used in combination with one another to limit and specify the search, and

another regular text search was performed without using MeSH or Emtree terms to include possible

recently published studies which had not been indexed in the MeSH or Emtree system. This did not retrieve

any new studies for this review. The exact search terms used in the two databases are given in Table 1.

Database Articles retrieved Search Terms (MeSH etc.)

Medline 211 MeSH terms: Exercise, Dietary supplements, Nitric

Oxide, Nitrates, Arginine, Citrulline

Text search: Beetroot, Ergogenic, Performance

Embase 338 Emtree terms: Exercise, Nitrate, Beetroot, Citrulline,

Arginine, Diet supplementation

Text search: Nitric Oxide, Ergogenic, performance

Total articles 549


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The database search yielded 549 studies of which 45 were duplicates, resulting in 504 unique publications

which were selected for screening based on their titles and abstracts. Screening for eligibility and relevance

was performed on the basis of the inclusion and exclusion criteria.

4.2. Selection criteria and selection of studies

On the basis of conforming to the inclusion and exclusion criteria, as presented in Table 2, studies were

required to have at least one trial with an NO-related substance (NO3, NO2, NO, beetroot, beetroot juice,

arginine or citrulline) and contain either one placebo or control group. Furthermore, this review only

included in vivo studies conducted on healthy individuals aged between 15 and 45 years, with interventions

taking place under normoxic conditions. Studies were not eligible for inclusion if the subjects were non-

human, non-aerobic or used additional supplements, and the trials had to apply a quantifiable

measurement of ergogenic enhancement that could be classified as including an aerobic limiting

component such as either a direct performance measurement (time trial or time to exhaustion) or an

indirect performance measurement (VO2submax, VO2 peak or threshold/efficiency). Finally, the studies had

to be original articles published in peer-reviewed journals. Other article types, such as reviews, book

sections or opinion articles were not included. If there was insufficient information to exclude a study on

the basis of abstract and title, the study was included for further screening of the articles' methods and

results by careful reading.

Table 2 - Inclusion and exclusion criteria

Inclusion criteria Exclusion criteria

NO-related supplement

Placebo or control group

Healthy individuals (15-45 years)

Exercise in normoxic conditions

In vivo

Non-human studies

Non-aerobic exercise

Trials employing the use of additional

supplements

Trials not employing a quantifiable measure

of exercise performance

Non-peer-reviewed journals

The screening revealed 32 articles which were closely read to determine whether they met the criteria for

inclusion and further evaluation. The reference lists of the articles were checked for further relevant

publications which yielded an additional study, bringing the number of eligible studies up to 33.


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A closer evaluation of these studies revealed nine publications that did not fulfil the criteria and a single

study that could not be retrieved, which excluded them from the review. Accordingly, 23 publications were

included in total. To view the selection flow in more detail, see Figure 2.

To our knowledge one additional study (40), related to the current topic of interest, was published after the

literature search for this review and this was not included due to the time limit.

4.3. PEDro scale

To measure the methodological quality of the included studies the Physiotherapy Evidence Database

(PEDro) scale was used for the systematic review. The PEDro scale has previously been shown to be a valid

(41) and reliable (42) tool for quality assessment.

The scale was modified and nine criteria were used due to the purpose and specificity of this particular

supplementation review. This meant that two criteria were excluded. These were “#6 -Blinding of

therapist” because no therapist was involved in the study designs, and “#9 - Intention to treat” because the

studies used small sample sizes with a controlled supplementation strategy that resulted in very low

dropout rates. Likewise, baseline measurements were only rated in accordance with the key outcome. By


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this it is meant that a time trial, for example, must have been measured in a similar pre-experimental time

trial before any supplementation was given to use as a baseline measurement. Since all subjects were

categorized as healthy individuals, the severity of the conditions was not included in the rating in criteria

#4. Furthermore, during the PEDro rating the term key outcome was defined as the key outcome for this

specific review purpose. The key outcome was defined as those outcomes which provide the primary

measure of the effectiveness or lack of effectiveness in terms of ergogenic effects of nitrate

supplementation. Otherwise, ratings were used according to the PEDro guidelines (43), and points were

only awarded when a criterion was clearly satisfied.

4.4. Data Extraction and presentation

In this review all data are presented as described by the authors. However, to present a better overview

different units representing the same parameter were, if possible, recalculated by the mean data to match

each other, but no further standard deviations were calculated. This was performed in fitness and dose

presentation where fitness was recalculated from VO2peak and weight to mL/O2/kg and dose was

recalculated from grams to mmol, as suggested by Lidder & Webb (44) and is therefore only presented as a

mean value. Where specific key outcomes were described, but not quantified by the authors, these results

are not included in the figures, but they are included in the summarizing table. Furthermore, some studies

included multiple key outcomes which are all included in this review and presented individually in the

figures. Accordingly, a single study can appear several times in a particular figure but the specific values are

described by the relevant variable.

To create an overview when comparing different parameters, these were divided into three subgroups, as

shown in Figure 3.

1. Subjects were divided by their training

status into trained and untrained, as

described by the authors.

2. The test interventions were divided into

direct performance measurements or

indirect performance measurements

relative to the parameter measured. To

qualify for a direct performance

measurement the measurement was

required to be either a time trial in terms

•Trained (as described ≈ >55 mL O2/kg/min)

•Untrained (as described ≈ <56 mL O2/kg/min) Traning status

•Direct Performance Measurement (Time trial or Time to exhaustion )

•Indirect Performance Messurement (VO2peak,VO2submax, Threshold/effeciency)

Exercise measurements

•Acute studies (≤1 day)

•Prolonged studies (>1 day) Supplementation

Figure 3 - Subgroup division


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of time or distance trial, or a time to exhaustion test. Other parameters were considered to be

indirect performance measurements, where VO2peak and peak power were classified relative to

the highest VO2 consumptions or power reached in the respective test. This means that a specific

VO2peak or peak power test was not necessarily performed, but was the highest measured in the

study. VO2submax was characterized as measuring oxygen consumption at a given submaximal

workload. The threshold/efficiency term includes data that can be characterized as either an

efficiency or threshold marker, combining values, for example from Power, Watt/VO2 ratios,

ventilator and/or lactate thresholds.

3. Finally, the supplementation strategies were divided into acute and prolonged studies, where acute

studies lasted no longer than a day and chronic studies lasted longer than a day.

In all the figures and tables the effects are presented as positive if they are considered ergogenic. Thus, an

increase in time to exhaustion and/or a VO2submax drop at a given intensity are ergogenic and are

therefore both presented as positive values. Effects are described as significant at P values below 0.05.

Where the parameters were compared, the effects are presented relative to the placebo group. If the

author(s) did not describe these relative values, they were recalculated by means of the absolute presented

values.

5. Results

5.1. General study characteristics

The individual data from the 23 selected articles are presented in Tables 3 and 4, which show the studies

and participant characteristics (reference number, author, design, PEDro score, sample size, age, fitness

status), dietary intervention (duration, form and dose) and exercise intervention and ergogenic effect

(exercise protocol, parameter and ergogenic effect).

In total 257 subjects were involved in the 23 included studies. Of these 226 were males, 14 were females,

and the sex of the other 17 subjects was not specified. Age ranged from 16 to 36 years. Most of the studies

(21 of 23) employed a crossover design, and 19 of these were also double-blinded. One study employed a

single-blinded approach, and another study did not blind its subjects. In total, 16 of the included studies

conducted randomized trials. The test protocols varied across the 23 studies, but 15 were conducted on

bikes, five evaluated supplementation during running, and three used a modified knee-extensor ergometer,

a combined arm and leg ergometer and a row ergometer, respectively.


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Table 3 - Summary of the included studies, describing their subjects as trained and examining the effect of nitrate supplementation on aerobic performance compared to a placebo

Study and participant characteristics Dietary intervention Exercise intervention and ergogenic effect

# Author Design PEDro Sample size

Age (years)

Fitness and description (mL O2/kg/min)

Duration Form and Dose

Type of work

Exercise protocol Parameter Ergogenic effect

(15) Christensen et al. 2013

CO, R 3 10 (men) 24 ± 4 72 ± 4 – Elite

6d BRJ – 8 mmol/day NO3

Bike 400 Kcal TT Submax (3*6min at 70%Wmax) 6*20s TT

TT VO2submax Mean power Peak power

→ → → →

(16) Peacock et al. 2012

DB, R 5 10 (men) 18 69.6 ± 5.1 – Elite

2.5h KNO3 – 9.9 mmol NO3

Run 5 km TT Submax (5min at 10 or 14 km/h)

TT VO2submax (10, 14km/h)

→ →

(23) Bescos et al. 2011

DB, CO, R 7 11 (men) 34.3 ± 4.8

65.1 ± 6.2 – Trained

3h NaNO3 - 0,16 mmol/kg NaNO3

Bike TTE (3.0W/kg +0.5W/Kg/min) Submax (4x6min at 2.0, 2.5, 3.0, 3.5 W/kg)

TTE VO2peak VO2/Power VO2submax (2.0, 2.5, 3.0, 3.5 W/kg)

→ ↓ ↑ →

(20) Wilkerson et al. 2012

SB, CO, R 5 8 (men) 31 ± 11 63 ± 7 – Trained

2,5h BRJ - 6.2 mmol NO3

Bike 50 miles TT TT VO2submax Power/VO2

→ → ↑

(17) Sunderland et al. 2011

DB, R 6 18 (men) 36.3 ± 7.9

61.7 ± 7.1 – Trained

28d L-arginine - 12 g/day

Bike TTE (50W+25W/min) VO2peak VT

→ →

(18) Bescos et al. 2012

DB, CO, R 7 13 (men) 32.6 ± 5.6

60 ± 7 – Trained

3d NaNO3 - 0,16 mmol NaNO3 /kg/day

Bike 40m TT TT VO2submax Mean Power

→ → →

(19) Cemark et al. 2012b

DB, CO 6 20 (men) 26 ± 1 60 ± 1 – Trained

1d BRJ - 8.7 mmol NO3

Bike 1h TT TT Mean Power

→ →

(13) Cermak et al. 2012a

DB, CO 7 12 (men) 31 ± 3 58 ± 2 – Trained

6d BRJ – 8 mmol/day NO3

Bike 10km TT Submax (2x30min at 45%, 60%Wmax)

TT Mean Power VO2submax (45%, 65% )

↑ ↑ ↑

(11) Lansley et al 2011b

DB, CO, R 6 9 (men) 21 ± 4 56.0 ± 5.7 – Trained

1d BRJ - 6,2 mmol NO3

Bike 16.1km TT 4km TT

TT 16.1km VO2submax (16.1km) Mean Power (16.1km) TT (4km) VO2submax (4km) Mean Power (4km)

↑ → ↑ ↑ → ↑

(2) Larsen et al. 2007

DB, CO, R 5 9 (men) 26 ± 6 55 ± 3.7 – Well-trained

3d NaNO3 - 0.1 mmol/kg/day

Bike VO2peak Submax (5x5min at 45, 60, 70, 80, 85 % VO2peak)

VO2peak VO2submax (Average)

→ ↑

(1) Bond et al. 2012 DB, CO, R 6 14 (men) 16.7 ± 0.5

Well-trained

6d BRJ - 5.5 mmol/day NO3

Rowing 6x500m TT TT (performance) ↑

Note: Design: DB: Double-blinded, SB: Single-blinded, CO: Cross-over, R: Randomized, B: Balanced. Form and dose: BRJ: Beetroot Juice, NaNO3: Sodium Nitrate, KNO3: Potassium nitrate, Exercise protocol and parameter: TT: Time Trial, TTE: Time to exhaustion, Submax: Submaximal intensity, GET: Gas Exchanges Threshold, VT: Ventilatory Threshold, Calculations: * calculated from VO2peak in l/min divided by mean weight


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Table 4 - Summary of the included studies, describing their subjects as untrained and examining the effect of nitrate supplementation on aerobic performance compared to a placebo Study and participant characteristics Dietary intervention Exercise intervention and ergogenic effects

# Author Design PEDro Sample size Age (years)

Fitness and description (mL O2/kg/min)

Duration Form and Dose

Type of work

Exercise protocol Parameter Ergogenic effect

(3) Larsen et al. 2011 DB, CO 6 14 (11 men, 3 women)

25 ± 1 56 ± 3 – Active

3d NaNO3 - 0,1 mmol/kg/day

Bike Submax (10min at 50% VO2max)

VO2submax Power/VO2

↑ ↑

(12) Lansley et al. 2011a

DB, CO 6 9 (men) 22 ± 4 55 ± 7 – Active

6d BRJ - 6,2 mmol/day NO3

Run/Walk TTE (GET+∆75% VO2max) Submax walk(10min at 4km/h), moderate run (6min at 80%GET)

TTE VO2peak VO2submax (walk, moderate run)

↑ ↓ ↑

(10) Koppo et al. 2009 DB, CO, R 7 7 (men) 21 ± 0.6 52.0 ± 4.8 – Active

14d L-arginine - 7,2 g/d

Bike Submax (6min at 80% VT) VO2submax

→

(22) Hickner et al. 2006

DB, CO, R

7 17 (undefined sex)

18 - 40 52.1 ± 1.9 – Active

1d L-citrulline – 3 g or 9 g

Run TTE (2mph+1mph/2min until 7mph afterwards incline 2%/1min)

TTE VO2peak

↓ →

(9) Wylie et al. 2013a DB, CO, R 6 14 (men) 22 ± 2 52 ± 7 – Recreationally active


Run YO-YO IR1 (20m shuttles runs with increasing speed)

TTE (Performance) ↑

(8) Bailey et al. 2009 DB, CO, R 7 8 (men) 26 ± 7 49 – Recreationally active

6d BRJ - 5,5 mmol/day NO3

Bike TTE (GET+∆70% VO2peak) Submax (20W+ until 80% GET)

TTE VO2peak VO2submax

↑ → ↑

(7) Bailey et al. 2010b

DB, CO, R 6 7 (men) 28 ± 7 47,9* – Recreationally active


Knee-extensions

TTE - High (30% MVC iEMG) Submax - Low (4min at 15%MVC iEMG),


↑ → ↑

(6) Bailey et al. 2010a

DB, CO, R 6 9 (men) 26 ± 6 47.0* – Recreationally active

3d l-arginine – 6 g/day

Bike TTE (GET+∆70% VO2max) Submax 1 and 2 (6min at 80% GET, 6m at GET+∆70% VO2max)

TTE VO2peak VO2submax (1, 2)

↑ → ↑

(5) Vanhatalo et al. 2010

DB, CO 5 8 (5 men, 3 women)

29 ± 6 46.8* – Healthy

2,5h, 5d, 15d

BRJ - 5.2 mmol/day NO3

Bike TTE (30W/min) Submax (5min at 90% GET)

VO2peak (2,5h, 5d, 15d) GET W (2,5h, 5d) GET W (15d) Peak Power (2,5h, 5 d) Peak Power (15d) VO2submax (2,5h, 5d, 15d)

→ → ↑ → ↑ ↑

(21) Larsen et al. 2010 DB, CO, R 5 9 (7men, 2 women)

30± 2.3

Healthy

3d 1h

NaNO3 - 0.1 mmol/kg/day NaNO3 - 0,33 mmol/kg

Arm and leg ergometer

TTE Submax (5min at 86±4W)


→ ↓ ↑

(4) Wylie et al. 2013b DB, CO, R 6 10 (men) 22±5 Healthy

2.5h BRJ - 4.2, 8.4 or 16.8 mmol NO3

Bike TTE (GET+∆75% VO2max) Submax (5min at 80% GET)

TTE (4.2) TTE (8.4, 16.8) VO2peak (4.2, 8.4, 16.8 ) Submax (4.2, 8.4) Submax (16,8)

→ ↑ → → ↑

(14) Murphy et al. 2011

DB, CO 6 11 (5 men, 6 women)

25±4 Recreationally fit

1.25h Baked BR – 8 mmol NO3

Run 5km TT TT (Performance) →

Note: Design: DB: Double-blinded, SB: Single-blinded, CO: Cross-over, R: Randomized, B: Balanced. Form and dose: BRJ: Beetroot Juice, NaNO3: Sodium Nitrate, KNO3: Potassium nitrate, Exercise protocol and parameter: TT: Time Trial, TTE: Time to exhaustion, Submax: Submaximal intensity, GET: Gas Exchanges Threshold, VT: Ventilatory Threshold, Calculations: * calculated from VO2peak in l/min divided by mean weight


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The majority of studies investigated more than one parameter when evaluating the ergogenic effects of

their supplementation strategy. These parameters were divided into direct and indirect performance

measurements with a relative distribution, as presented in Table 5. The outcomes of the direct

performance measurements (time trial and time to exhaustion) were included in 18 of the studies and had

a total of 21 unique measurements in contrast to the indirect performance measurements (VO2peak,

VO2submax and thresholds/efficiency) which were typically present multiple times in the studies, providing

a total of 57 quantified outcomes and included in 21 of the 23 studies.

5.2. Study Quality

The modified PEDro scale (with a maximum score of 9) was used to rate the quality of the included studies.

As shown in Table 6 the studies consistently scored between five and seven points, apart from one study,

which scored only three points. The PEDro scale ratings allocated studies points for three criteria: random

allocation of the subjects to their groups (#2), between-group statistical comparisons (#10) and measures

of variability (#11). Likewise, the studies generally showed a strong tendency to blind the subjects (#5) and

the assessors (#7) with 21 and 19 studies, respectively, employing this approach. Regarding measuring

criterion #8, at least one key outcome was obtained from over 85% of the initially allocated subjects where

15 of the 23 studies scored points for this criterion.

Direct Performance Measurements Indirect Performance Measurements

Time trials:

9 unique studies

10 quantified outcomes Time to exhaustion:

9 unique studies

11 quantified outcomes

VO2peak:

11 unique studies

14 quantified outcomes VO2submax:

17 unique studies

27 quantified outcomes Thresholds/efficiency:

11 unique studies

16 quantified outcomes

Table 5 - Quantification of Direct and Indirect Performance Measurements


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Table 6 - PEDro rating of included studies

Study Criteria

# Author

#1 Specified eligibility criteria

#2 Randomized allocation

#3 Concealed allocation

#4 Baseline group similarity

#5 Blinding of subjects

#6 Blinding of therapists

#7 Blinding of assessors

#8 At least 85% subject measurements

#9 Intention to treat

#10 Between-group comparisons

#11 Point and variability measures

Total

(8) Bailey et al. 2009 1 1 1 - 1 1 - 1 1 7

(6) Bailey et al. 2010a 1 1 - 1 1 - 1 1 6

(7) Bailey et al. 2010b 1 1 - 1 1 - 1 1 6

(23) Bescos et al. 2011 1 1 1 - 1 1 - 1 1 7

(18) Bescos et al. 2012 1 1 1 - 1 1 - 1 1 7

(1) Bond et al. 2012 1 1 1 - 1 - 1 1 6

(13) Cemark et al. 2012a 1 1 1 - 1 1 - 1 1 7

(19) Cermak et al. 2012b 1 1 - 1 1 - 1 1 6

(15) Christensen et al. 2013 1 - - 1 1 3

(22) Hickner et al. 2006 1 1 1 - 1 1 - 1 1 7

(10) Koppo et al. 2009 1 1 1 - 1 1 - 1 1 7

(12) Lansley et al. 2011a 1 1 1 - 1 - 1 1 6

(11) Lansley et al. 2011b 1 1 1 - 1 - 1 1 6

(2) Larsen et al. 2007 1 1 - 1 - 1 1 5

(21) Larsen et al. 2010 1 1 - 1 - 1 1 5

(3) Larsen et al. 2011 1 1 - 1 1 - 1 1 6

(14) Murphy et al. 2011 1 1 - 1 1 - 1 1 6

(16) Peacock O et al. 2012 1 1 - 1 - 1 1 5

(17) Sunderland et al. 2011 1 1 1 - 1 - 1 1 6

(5) Vanhatalo et al. 2010 1 1 - 1 - 1 1 5

(20) Wilkerson et al. 2012 1 1 1 - - 1 1 5

(9) Wylie et al. 2013a 1 1 - 1 1 - 1 1 6

(4) Wylie et al. 2013b 1 1 - 1 1 - 1 1 6


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Data from the PEDro scale ratings showed that none of the included studies scored points for concealed

allocation (#3) in their selection of subjects. Equally, the studies generally revealed a lack of quality

concerning eligibility criteria (#1) specified in the studies, with studies describing their subjects but not

presenting any criteria for inclusion of these. Similarly, only five studies compared the subjects in terms of

key outcome at baseline (#4), whereas most of the studies employed a baseline measurement under rest

conditions, which, in this case, did not fulfil the criterion.

5.2.1. Summary

With regard to quality, the studies achieved relatively homogeneous scores of between five and seven

points, with only one study receiving a lower score. The studies' strength in blinding indicates reasonably

valid studies which primarily lacked concealed allocation, baseline group similarity, and specified eligibility

criteria, which could have influenced the outcome and is indicative of the variety of methodologies.

5.3. Training status

The training status of the participants was highly variable across the included studies, with fitness values

ranging from 46.8 mL O2/kg/min to 72 ± 4 mL O2/kg/min, while four studies do not describe any fitness

measures. Of the 23 included studies 11 describe their subjects as trained, well-trained or elite with a

fitness value of >55 mL O2/kg/min, whereas one of the studies does not report an absolute fitness value.

The remaining 12 studies describe their subjects as recreationally fit, healthy or physically active,

presenting fitness values of <56 mL O2/kg/min, while three studies do not report an absolute fitness value.

As presented in Table 3 and illustrated in Figures 4 and 5, the ergogenic effects seem to be dependent on

level of fitness. Having observed the trained subjects, five of eleven studies do not report ergogenic effects

in any of their measured parameters, and further analysis of the seven studies with the highest fitness level

>60 mL O2/kg/min shows that none of these consistently reported an improvement after NO-related

supplementation. Only two of these seven (20, 23) studies propose an ergogenic effect in a single of several

measured outcomes. While one of the studies shows an improvement in the power/VO2 ratio, the other

shows improvement in the VO2/power ratio, but likewise observes a decrease in VO2peak which can be

described as an anti-ergogenic effect. The remaining four studies with fitness values <60 mL O2/kg/min

generally present results of an ergogenic effect, and all of them report an effect in one or more of the

following parameters: time trial, VO2submax, mean power and VO2peak. However, it is still worth

mentioning that as described above only one of the 11 studies concerning trained subjects shows a

negative effect of supplementation in terms of a decrease in VO2peak, yet another study tends to see a

decrease as well, which is considered anti-ergogenic.


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In the untrained subjects improved exercise efficiency is reported relatively consistently for several

parameters, whereas a drop in VO2submax at a given intensity is the most prominent observed effect, as

eight of nine studies describe this. Likewise, seven measured outcomes out of a total of 11 favours an

ergogenic effect on either time to exhaustion or time trial, while only a single study (22) reports a

significant negative effect after supplementation. The results for VO2peak, are relatively reliable, as 12 of 14

outcomes did not differ significantly from the placebo, while two studies (12, 21) observed a decrease.

Regarding total measurements only two (10, 14) of the untrained studies do not present any significant

measurements in favour of NO-related measurements compared to placebo, and one of these two studies

(14) measured a 5 km running time trial, where performance tended to increase significantly after

supplementation with an increase in running velocity from 11.9 ± 2.6 km/h to 12.3 ± 2.7 km/h (P=0.06).

5.3.1. Summary

These preliminary findings suggest that the ergogenic effect of NO-related supplementation is dependent

on the individual’s fitness level, as more promising results were observed in untrained individuals with a

fitness level <60 mL O2/kg/min, particularly in relation to time trials, time to exhaustion and VO2submax

measurements.

5.4. Exercise measurements, duration and intensity

In the included studies exercise performance was examined in multiple ways. The results are presented in

Tables 3 and 4, respectively divided into direct or indirect performance measurements’ ergogenic

outcomes. The studies typically included more than one outcome and as well as both a direct and indirect

performance measurement. However, while the majority of studies clearly yielded data during prolonged

or submaximal work, it is noteworthy that three studies included intense intermittent work.

5.4.1. Direct performance measurements

From a direct performance measurement perspective a total of 21 outcomes are presented, 11 of these

favouring an ergogenic effect of NO-related supplementation. In contrast, nine studies found no effect, and

only one study (22), evaluating time to exhaustion, observed a significant decrease in performance, by 0.8

% (p<0.05).

The time to exhaustion results are presented in Figure 6 which shows seven of 11 studies favouring

supplementation with improvements ranging from 4.2% to 28.8%, with the majority of six studies showing

improvements above 11%. Three studies reported positive changes from 1.7% to 8.1%, which were not


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significantly different from placebo. The last study as presented above found a significant decrease in time

to exhaustion with a reduction of 0.8%.

The time trial, presented in Figure 7 shows notably smaller changes than the time to exhaustion results,

with a total of 10 outcomes being presented. Four of these reveal a significant ergogenic effect with

changes ranging from 1.24% to a maximum of 2.89%. However, notably a single study by Murphy et al. (14)

reveals a tendency to be significant with an increase of 3% (P=0.06). Finally, in the remaing six studies no

effect was seen, but in one study by Peacock et al. (16) there was a tendency for a slight anti-ergogenic

effect of 0.9% (P=0.12).

5.4.2. Indirect performance measurements

In terms of indirect performance measurements only two studies did not include any, whereas the

remaining studies generally measured multiple indirect performance measurements. Figure 8 shows a total

of 14 measurements of VO2peak relative changes presented by the respective authors. Three studies (12,

21, 23) found decreases in VO2peak of -5.7%, -3.7% and -2.7%, respectively and two additional studies (2,

7) likewise tended to see this effect, with insignificant mean VO2peak decreases of -4.6% and -2.6%

respectively. Three studies show insignificant results displaying both positive and negative mean values

ranging from -5.7% to an increase of 2.3% which tended to be significant. Furthermore, one study (22) did

not specify the data, but described the results as being not significant.

The threshold/efficiency measurements, presented in Figure 9, were investigated according to 16

outcomes, and included measurements of power, thresholds and calculated ratio, as described earlier. In

total, seven of these presented significant positive effects of supplementation ranging from increases of

2.1% to 25%. However, nine studies did not find any significant effects, and a further two studies (5, 19)

revealed effects indicating a decrease in efficiency of 1% and 5% respectively.

A relatively consistent finding is a reduction in VO2submax at a given workload, indicating an improvement

in economy of work, as presented in Figure 10, where 14 of 27 outcomes show such an improvement after

supplementation with an NO-related supplement. The remaining studies found no significant effect, but all

tended towards positive effects, apart from a single study by Koppo et al. (10). The combined results of

relative changes in VO2submax range from -0.23% to 25.2%, with significant studies starting at 3%. The

largest changes were found by Bailey et al. (7) who reported an increase of 25.2%. However, contrary to

the remaining studies evaluating bike and running exercise protocols, it is noteworthy that his study was

conducted with a leg extension ergometer which therefore yielded small absolute VO2 values of 484 ± 41


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and 362 ± 30 mL VO2/min, respectively, for placebo and NO3. Likewise, Lansley et al. (12) found another

large difference with an 11% relative change, although this was when walking. However, the majority of

studies ranged from changes of 3% to 7%.

5.4.3. Duration and intensity

The majority of studies included in this review, presented in Tables 3 and 4 investigated either short direct

or indirect performance measurements. From an indirect perspective the submaximal assessments

generally lasted around 4-6 minutes with a single study by Cermak et al. (19) measuring VO2 during 30

minutes of exercise. The direct measurements of time to exhaustion trials naturally had a higher intensity,

especially in the final stages of the test, and the duration was typically shorter, lasting around 8 minutes,

with a single study by Hickner et al. (22) lasting 15 minutes. On the contrary, the time trials were more

prolonged in duration and, accordingly, the intensity was somewhat lower, whereas the duration lasted

from 6 minutes to 137 minutes. However, only three trials, by Wilkerson et al. (41), Bescos et al. (18) and

Cermak et al. (19) tried to assess longer trials (≥40 min) and in these there was no significant performance

enhancement in contrast to the shorter trials (≤40 min) by Cermak et al. (13), Lansley et al. (11) and Bond et

al. (1).

5.4.4. Summary

In relation to the different exercise types and intensities the existing studies generally imply results

favouring performance improvements. Of these, the ergogenic effect in direct performance measurements

seems larger in time to exhaustion than time trials, and in terms of indirect performance measurements

VO2submax seems more consistent than threshold/efficiency, whereas the data on VO2peak seem more

inconsistent. In the included studies duration is typically short while intensity varies.


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Figure 4 - Relative effects on direct performance measurements (time trials and time to exhaustion) in relation to the subjects’ mean fitness (mL O2/kg/min) after NO-related supplementation relative to the control group. Grey filled: significant changes, White filled: non-significant changes. P˂0.05.

Figure 5 - Relative effects on VO2submax in relation to the subjects’ mean fitness (mL O2/kg/min) after NO-related supplementation relative to the control group. Grey filled: significant changes, White filled: non-significant changes. P˂0.05.

Figure 6 - Relative effects on time to exhaustion after NO-related supplementation in relation to the control group. Grey filled: significant changes, White filled: non-significant changes. P<0.05.

Figure 7 - Relative improvements in time trials after NO-related supplementation in relation to the control group. Increases suggest a drop in time trial performance. Grey filled: significant changes, White filled: non-significant changes. P<0.05.


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Figure 8 - Relative effects on VO2peak after NO-related supplementation in relation to the control group. Grey filled: significant changes, White filled: non-significant changes. P<0.05.

Figure 9 - Relative effects on threshold/efficiency after NO-related supplementation in relation to the control group. Grey filled: significant changes, White filled: non-significant changes. P<0.05.

Figure 10 - Relative improvements in VO2submax after NO-related supplementation in relation to the control group. Increases suggest a drop in VO2submax at a given workload. Grey filled: significant changes, White filled: non-significant changes. P<0.05.


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Figure 11 - Relative effects on nitrite after NO-related supplementation in relation to the control group. Grey filled: significant changes, White filled: non-significant changes. P<0.05.


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5.5. Supplement, timing and quantity

Multiple supplementation strategies were used in the studies reported, with the majority of studies (19 of

23) using supplements targeting the NOS-independent pathway of NO synthesis. The most commonly used

supplement in these studies was beetroot juice, with 12 of the 19 studies using this approach. Five studies

used sodium nitrate as a substrate, and the two least used supplements was potassium nitrate and baked

beetroot, which just one study used respectively. Only four of the included studies in this review employed

supplements targeting the NOS-dependent NO synthesis pathways. Three studies used arginine and a single

study used citrulline for supplementation to evaluate possible performance effects.

To quantify the effect of the supplements most studies describe changes in plasma NO2 concentration after

ingestion, as presented in Figure 11. Reviewing these results, it is clear that supplementation significantly

changed the plasma concentration of NO2, ranging from 21% to 377% relative to placebo. While 11 of the

17 measurements reported NO2 levels in a range between 50% and 150%, four studies reported an even

greater effect, with an increase of between 218% and 377%. These high levels seem dependent on the

amount of NO3 ingested, as three of these four studies supplemented with levels above 8.4 mmol NO3/day.

While only studies reporting NO2 plasma concentrations are presented in the graph, other studies

measured the supplementation effect as predictors of NO production, arginine levels, NO3 levels, urinary

creatinine or an addition of several NO metabolites (NOx). Two studies found no effect on NO plasma

concentration parameters after supplementation prior to the testing. One of the studies used citrulline and

the other used arginine as a supplement. Another arginine study found changes in arginine, but not in NO-

related levels.

The timing of supplementation varied from 1 hour to 28 days. Therefore, the duration was divided into

acute studies supplementing <1 day before performance and prolonged studies with a duration longer than

a day. Nine studies employed an acute supplementation design, with five studies timing intake 2.5 hours

prior to the exercise intervention, two studies ingesting 3 hours prior to the exercise intervention and two

studies ingesting baked beetroot 1 hour and 15 minutes before and sodium nitrate 1 hour before the

exercise intervention, respectively. 15 studies used a prolonged approach, the longest supplementation

period being with citrulline lasting 28 days. In the prolonged supplementation studies the timing ranged

from 1 to 28 days, with one study evaluating 2 days, five studies 3 days and six studies 6 days. The three

remaining studies evaluated supplementation exceeding 6 days, at 14, 15 and 28 days respectively. In this

review only one study by Vanhatalo et al. (5) compared the effects of the same dose of NO3 using both an

acute and prolonged approach, and another study by Larsen et al. (21) examined both acute and prolonged


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supplementation, but with different doses, and it is therefore included in both strategies. The results of the

various supplementation strategies applied in the existing literature, presented in Tables 3 and 4, illustrate

that acute and prolonged studies both seem to induce effects in performance with an effect being

observed in six out of ten acute studies and ten out of 15 prolonged studies.

From a quantity perspective the NO3 doses were given using two different methods, whereas four studies

used a relative dose consisting of NO3 quantities ranging from 0.01-0.02 mmol/kg. Meanwhile, the absolute

doses of NO3-related substance ranged from 5.1 mmol/dose to 16.4 mmol/dose. In these studies the

majority of interventions (12 out of 19) used doses in the 5-8mmol NO3/dose domain. The three

interventions using arginine as a supplement were in two studies using 6-7 g/day, whereas a single study

used 12 g/day. The citrulline intervention compared ergogenic effects of 3 grams ingested 3 hours prior to

testing or 9 grams ingested during a 24-hour period. Two studies examined the direct effect of different

doses of supplement.

5.5.1. Summary

The preliminary findings on supplementation strategy suggest that the most common supplement

substrate used was beetroot juice which both acute and prolonged studies targeted to induce changes in

plasma NO2 content. Furthermore, it seems that larger doses have a greater NO2 plasma response.

5.6. Overall results summary

To summarise the results, 12 of the 23 included studies favoured supplementation as a result of ergogenic

improvements in one or more measured parameters, while seven studies found no effect after

supplementation. Three studies observed both ergogenic and anti-ergogenic effects, and a single study

found a negative effect on performance after supplementation.

6. Discussion

As presented in Tables 3 and 4 and Figures 4 and 5 a clear effect of NO-related supplementation is not

consistently evident. However, from a broad perspective it seems that a few parameters are likely to

improve as a result of supplementation. Nevertheless, the controversy remains in relation to individual

training status, exercise measurements, duration, intensity and the supplementation strategy employed.

6.1. Training status

On the basis of the literature some researchers (13, 15-17, 19, 20) have suggested an interaction between

training status and the ergogenic effect of NO-related supplementation. This seems very likely since the


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results from this review indicate several ergogenic effects for the majority of untrained subjects while the

ergogenic effects seem less obvious in better trained subjects. There is no doubt that the significant

methodological differences between the studies, especially concerning duration, form of supplementation

and exercise protocol make them difficult to compare. Moreover, the question remains where this gap in

effects occurs since some studies, which describe their athletes as trained, found a performance enhancing

effect and better exercise efficiency after nitrate supplementation, while this effect was not seen in other

studies involving better trained or elite individuals. Jones et al. (45) argues that the great difference in the

physiological training adaptions of untrained versus trained individuals can be explained by two main

factors: high NOS activity and/or less hypoxia development.

6.1.1. NOS activity in trained subjects

Christensen et al. (15) speculate that due to highly trained subjects’ long-term endurance training

adaptations they already have optimal NO synthetic capabilities, producing enough NO for aerobic

performance which contrasts with the moderately trained subjects. This could interfere with the effect of

NO supplementation limiting the ergogenic results in highly trained subjects. Wilkerson et al. (20) argue

that well trained athletes have higher NOS activity so that the NOS-independent pathway may be relatively

less important for the generation of NO, thereby accounting for the inconclusive results. Furthermore, this

would result in what Christensen et al. (15) and Bescos et al. (23) propose and describe, namely that

aerobically fit subjects have high basal levels of plasma NO3 and NO2. This is confirmed by Wilkerson et al.

(20) who present data that show that endurance trained subjects have a higher NO2 pre-supplementation

baseline compared to untrained individuals. However, on the contrary, Peacock et al. (16) observed lower

initial NO3 and NO2 values at pre-supplementation in cross-country skiers, than other studies have

observed. Peacock et al. (16) could not explain the discrepancy of this observation, but argued that lower

NO3 concentration may exist in different populations, which needs to be addressed in future research.

However, the suggested high basal NO3 or NO2 plasma concentration in highly trained athletes could

likewise result in a lower NO2 response when receiving a supplement. Peacock et al. (16) support this

hypothesis by suggesting that untrained subjects reach a higher NO2 level after NO3 supplementation than

highly trained subjects. Likewise, Cermak et al. (19) argue that the inconclusive ergogenic results in well

trained athletes are related to the subjects’ NO2 response and propose that low or lack of ergogenic effects

is connected to no increase in plasma NO2. Reviewing trained subjects in studies connected to the reported

NO2 response, as presented in Figure 11, the majority of trained subjects did not yield a relatively large NO2

response compared to untrained individuals, which supports the hypothesis of a lower NO2 response after

supplementation.


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However, given the possibly larger NO3 and/or NO2 baseline values and a a relatively lower NO2 response in

highly trained subjects, as Bescos et al. (18) propose, these subjects may need a higher dose or longer

supplementation to elicit an effect. Supporting this, Wilkerson et al. (20) noted a correlation between the

increase in plasma NO2 and performance, and proposed that beetroot juice supplementation still seems to

be ergogenic in well trained subjects if the NO2 level is sufficiently elevated (R=0.83, P=0.01).

6.1.2. Development of Hypoxia

The lower effect of NO-related supplementation in trained subjects compared to untrained subjects may

also be explained by lower hypoxia, and the development of acidosis during exercise in highly trained

subjects reduces reliance on NO synthesis through the reduction of NO2. Wilkerson et al. (20) explains that

this phenomenon is likely to be due to highly trained subjects having greater mitochondrial and capillary

density. This may limit the development of hypoxia and acidosis in skeletal muscles during exercise, by

improving transit time for oxygen offloading, thereby preserving NOS function and reducing the

contribution of the NOS-independent reduction from NO3 to NO. If subjects do not experience the same

hypoxia and acidosis, they would not have the same ability to convert the ingested NO-related supplement

to NO, and this would be obvious in a lack of NO2 decline during exercise. A few of the authors, including

Wylie et al. (9) and Peacock et al. (16), who have been studying the NO3/NO2 response to exercise have

observed a lack of decrease during exercise and suggest that this could be the reason for not observing an

ergogenic effect. Wylie et al. (9), in particular, found that only during the exhaustive test did their subjects

show a decrease in NO2 compared to the submaximal test. Due to the fact that NO2 seems to decline during

intensive exercise it is worth considering that a continued high elevation of NO2 may be advantageous and

this could give rise to speculation of further NO-related supplementation during competition.

6.1.3. Summary

It generally seems evident that untrained subjects benefit from NO-related supplementation. However, it

remains relatively unclear if NO-related supplementation is beneficial in highly trained athletes.

Nevertheless, due to the lack of statistical significance in elite groups, it still cannot be concluded that NO-

related supplementation does not have an effect. It is worth mentioning that in an elite sport context even

the smallest positive effect on performance or exercise efficiency may be of great importance. These

combined results suggest that a sufficient elevation in NO2 is needed to induce an effect, and some studies

have proposed that trained athletes may need a higher dose or longer supplementation period to induce a

beneficial effect. However, no study specifically addresses this hypothesis and thus it remains unproven.


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Finally, it should be noted that some studies show a possibility of a decrease in VO2peak after NO-related

supplementation even though performance does not seem to be affected by this reduction.

6.2. Exercise measurements, duration and intensity

Performance-enhancing effects in interaction with the type of exercise measurement, duration and

intensity have not been clearly documented, and the current literature uses various tests and work

conditions to assess knowledge of NO-related supplements' effect on performance, which yield

inconsistent results.

6.2.1. Direct performance measurements

From a direct performance measurement perspective, seven of the 11 studies evaluating time to

exhaustion tests favoured NO-related supplementation over placebo, and two (4, 21) of the remaining four

studies had results tending towards increased performance. On the contrary, only four out of ten time trials

favoured NO-related supplementation over placebo, and none of the remaining time trial results tended to

show improved performance. This is somewhat in agreement with Hoon et al.’s (46) meta-analysis which

revealed an ergogenic effect in three time to exhaustion studies with a pooled effect size of 0.79 (0.23 to

1.35, P=0.006). However, Hoon et al. (46) observed a small but non-significant ergogenic effect of NO3

supplementation in pooled effect size in time trials, of 0.11 (95 % CI: -0.16 to 0.37, P=0.43). Having divided

their studies into graded exercise tests, they did not find any significant effect either, with an effect size of

0.26 (-0.10 to 0.62, P=0.16). Furthermore, the relative improvements in time to exhaustion results seem

larger that the time trial results, which are also presented in another review by Jones et al. (47). The

discrepancy in the lower number of significant results and relative changes in time trials could be

associated with what Lansley et al. (11, 12) and Cermak et al. (13) found, that time trials are thought to be a

more ecologically valid measurement due to the fact that time to exhaustion is not present in any typical

real life athletic aerobic performance. Time trials are therefore proposed to be a more accurate simulation

of physical response during competition and actual race performance as they resemble the demands of a

competition. Given this, Hopkins et al. (48) indicate that improvements in time trials are far smaller,

whereas a 20% improvement in time to exhaustion would be expected to correspond to 1–2% in time trial

performance.

6.2.2. Indirect performance measurements

A review of the results of a decrease in VO2peak would initially be assumed to be an anti-ergogenic effect.

However, in all of these studies work rate and performance did not decrease. In fact, the three studies (12,

21, 23) showing a significant decrease in VO2peak actually found results that favour other ergogenic


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improvements, whereas Larsen et al. (21), who saw a VO2peak drop of 2.6%, observed the trend in time to

exhaustion to increase, and Bescos et al. (23) actually saw an improved VO2/power ratio during the exercise

protocol despite seeing a 3.7% VO2peak drop. In contrast to these two studies, Lansley et al. (12) found a

reduction of 5.7% in VO2, but likewise reported a time to exhaustion improvement of 15%. These combined

results suggest that the enhanced effect of NO is sufficient to offset the reduced VO2peak, and Bescos et al.

(23) and Larsen et al. (21) suggest that the VO2peak decrease may be explained not by a decrease in non-

ATP production but O2 consuming functions in the muscle tissue e.g. less proton leakage. However, there is

insufficient data to draw any conclusions about this. In contrast to the studies discussed above, Vanhatalo

et al. (5) present data which show a tendency for a small increase in VO2peak after 15 days of NO3

supplementation. They explain this effect by NO-mediated effects that could be due to prolonged

supplementation, which could include mitochondria biogenesis. This is different from the above-mentioned

studies which applied shorter supplementation strategies for a maximum of 6 days. However, there is yet,

insufficient evidence to accept this hypothesis.

In terms of VO2submax two studies reported much greater effects than the remaining study, with 11.5%

and 25.2% increases, respectively. This could be attributed to the fact that Lansley et al. (12) studied

performance while walking, and Bailey et al. (7) used a leg extension ergometer which yield lower absolute

VO2 values (770 mL/min and 389 ± 16 mL/min) compared to higher intensity aerobic performance.

Supporting this Lansley et al. (12) observed that when intensity increased, the relative effect declined,

whereas walking improved VO2submax by 12%, moderate running by 7% and the VO2peak during intense

running by 5%. This is confirmed by Bailey et al. (7) who in a study employing leg extension with a small

workload (15% MVC in 4 min), found a 25% drop in VO2submax compared to placebo. Relatively consistent

VO2submax improvements of NO-related supplementation are likewise reported by eight out of 11 studies,

which observed improvements in direct performance measurements in time trials or time to exhaustion.

As with VO2submax the threshold/efficiency data seem somewhat consistent with an improvement after

supplementation, as presented in Figure 9. A study with a great impact on threshold/efficiency was

conducted by Vanhatalo et al. (5). Their study found both the largest decrease in the gas exchange ratio,

with a change of -5 % after 2.5 hours of supplementation, and the largest increases of 13% and 25%, after 5

and 15 days of supplementation respectively. These measurements are presented as work rates in Watts.

However, Vanhatalo et al. (5) found no changes in VO2 at any of these thresholds. They offer no clear

explanation for this but try to put this data in perspective by stating that longer supplementation may be

needed to induce changes in VO2.


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6.2.3. Duration and intensity

The relatively consistent reduction in VO2submax and threshold/efficiency measurements indicates an

improvement in economy of work which would typically be transferable to a performance enhancement in

prolonged lower intensity exercise performed (49). However, some authors and recently published reviews

(13, 21, 46, 47) have suggested that the magnitude of effects is somewhat dependent on the intensity and

duration of exercise. Relative short, high-intensity exercise until 15 minutes has been confirmed as

ergogenic when supplemented with NO-related substance in some of the included studies as shown by e.g.

Lansley et al. (11) and by Cermak et al. (13). Whereas Lansley et al. (11) state, that this indicates that an

effect is dependent on a time of between 5 and 30 minutes. The hypothesis for this time-dependent

relationship is also described by Wilkerson et al. (20) who argue that lower-intensity exercise targets the

aerobic system to provide the energy, and therefore may not fully elicit the independent pathway (13, 18).

Meanwhile, as already stated, Wilkerson et al. (20) did not find any improvement in a longer, 50 mile, time

trial (≈137 min) which was performed at relatively low intensity (≈75 % VO2max). This may be explained by

the fact that their subjects were highly trained, and/or the low intensity which may have limited the

independent pathway due to the weak development of acidosis and hypoxia. Likewise, Bescos et al. (18) in

2012 found no significant effect after a 40 minute time trial. Shorter, more intense work may result in more

pronounced independent pathway activation by increasing intracellular acidosis which would potentiate a

reduction of NO. This is in good agreement with the time to exhaustion measurements included, which

were typically achieved from protocols that last around 5 minutes, giving the exercise protocol result of

high acidosis and hypoxia. Likewise, in this review shorter high intensity time trials were performed in some

studies. For example, Cermak et al. (13) and Lansley et al. (11) both observed an improvement in 4.1 km, 10

km and 16.1 km cycling time trial performances after NO-related supplementation compared to placebo.

However, Murphy et al. (14) only saw an tendency for improvement in 5 km run time trials (P=0.06) but did,

however, observe a significant effect in the final 1.8 km of the 5 km time trial compared with placebo

(P=0.04). This apparent effect in the last part of a protocol was also described by Christensen et al. (15) in

their prolonged study, which presents results showing that the subjects completed the last 10 miles of the

50-mile time trial faster after supplementation whereas the acid and hypoxic environment seemed to be

more pronounced than during the initial 40 miles. Similar findings were observed in higher intensity

intermittent work. Bond et al. (1) saw a smaller but still significant performance-enhancing effect of 0.40%

in very well trained rowers tested in 6x500 m all-out efforts, but more interestingly, they also observed that

the greater improvement was observed particularly in the fourth through sixth repetitions, and they argued

that this was due to an increase in hypoxic and acidosis conditions. This is supported somewhat by another


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high intensity intermittent study by Wylie et al. (9), who observed an improved performance of 4.2% in

intense intermittent exercise performed by recreational soccer players. To investigate this they used a YO-

YO IR1 interval test. This is a progressive shuttle test that has been seen to be correlated with performance

and assesses both the aerobic and anaerobic energy systems, especially in the final stages before

exhaustion (50). However, investigating more extreme intense work Christensen et al. (15) did not report

any effect on peak or mean power during 6x20 second bike sprints with a 100 second rest, after 6 days of

supplementation, ultimately suggesting that the NO-related supplementation effect is not apparent in high

intensity work targeting the creatine phosphate ATP synthesis pathway.

6.2.4. Summary

These results suggest that NO-related supplementation has performance-enhancing effects in both low

submaximal and higher intensity exercise. The scientific foundation is, however, still somewhat unclear as

to whether longer low intensity or shorter high intensity exercise is more responsive to NO-related

supplementation. It may be speculated that low intensity exercise does not fully elicit the potential for an

NO-related ergogenic effect. However, improvements in VO2submax have been consistently confirmed,

which supports improvements in longer trials.

6.3. Supplement, timing and quantity

There was wide variation in the supplement strategies in the included studies and in the manner in which

NO was employed, using both independent and dependent forms of substrates.

6.3.1. Supplement

Some authors, including Bloomer et al. (51) and Sureda & Pons (52) proposed that independent pathway is

superior to the NOS dependent pathway of NO synthesis during exercise. Given the fact that subjects

experience hypoxic circumstances the body’s demand for oxygen simply cannot be met and other co-

factors may limit the NO synthesis by the dependent pathway citrulline and arginine. This hypothesis is

supported to some extent in this review. Whereas the independent substance induces the NO2 response

and this is generally translated into performance enhancement, only one of the three arginine studies

found an increase in NO production, and this is also the only one that observed improvements in time to

exhaustion and VO2submax. These speculations are supported by recent studies by Alvares et al. (53),

Vanhatalo et al. (54) and Forbes & Bell (55) which did not see a rise in NO after arginine supplementation.

The formation of NO after supplementation with arginine seems dependent on other factors than simply

ingestion of arginine. Since the increase in NO after arginine supplementation has been questioned, there

has recently been increasing interest in citrulline supplements. This is thought to be due to the fact that


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arginine is proposed by Hartman et al. (56) to be subject to extensive pre-systemic and systemic elimination

by arginine in the gut wall and liver, whereas citrulline does not encounter this pre-systemic elimination.

Hartman et al. (56) therefore speculated that ingestion of citrulline is a more effective way to increase NO

levels than arginine supplementation. However, this effect was not present in the included studies of which

only one, by Hickner et al. (22), used citrulline which controversially did not find any difference in plasma

NO metabolites before exercise, which may explain the direct negative effect, in terms of a lower time to

exhaustion after supplementation compared with placebo. Sureda & Pons (52) proposed that most

citrulline interventions have been conducted using a combination with malate which may increase levels of

NO metabolites and thereby enhance performance. These interventions were excluded because this review

only considers supplementation without any other active ingredient, limiting the number of citrulline and

arginine studies and thus there is no conclusive evidence on these supplements. To summarize, the

independent substance seems effective in inducing changes in plasma measurements and performance.

6.3.2. Timing and quantity

As implied above, an ergogenic effect seems to be somewhat related to high NO2 levels, and several

authors correlate the changes in plasma NO2 to changes in performance (9, 20). Recent reviews by

Dreissigacker et al. (57) and Rassaf et al. (58) have likewise identified plasma NO2 as an important element

of exercise tolerance in healthy untrained individuals, whereas Totzeck et al. (59) also found a similar

correlation in highly trained athletes between a high endogen level of NO2 and a superior work capacity.

Even though these correlations have been observed, there is still not yet a clear quantity and timing

relationship. From a timing perspective the ingestion of NO3 was found by Vanhatalo et al. (5) to increase

NO3 levels rapidly after approximately 30 minutes, peaking 1.5 hours later. Likewise, Wylie et al. (4) found a

peak elevation in NO3, which occurred 1 hour post administration for 4.2 and 8.4 mmol NO3 and 2 hours

post administration for 16.8 mmol NO3, respectively suggesting that this effect should be attainable from 1

hour after ingestion depending on the amount of NO3 ingested. Supporting this, Larsen et al. (21) found a

significant decrease in VO2 at submaximal intensity, 1.45 ± 0.08 to 1.37 ± 0.09 L/min (P<0.05) 1 hour after

ingestion of NO3. However, not all NO3 is reduced to NO2. This process takes further time, which implies

that the elevation in NO2 is typically seen peaking within 2-4 hours with a concentration increase about two

to threefold, also shown by Wylie et al. (4). Given the fact that acute studies see either an improvement in

performance or in exercise efficiency (14, 21) arising from NO3 doses up to 1 hour before exercise, it is

suggested that effects may occur within a relatively short time frame and may not need to be

supplemented for a prolonged period.


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However, the question remains about what effect prolonged NO-related supplementation provides, which

has been addressed in a recently published review by Hoon et al. (46), who imply that a multiple day dosing

strategy may be more efficacious for improving exercise performance than acute strategies. Reviewing

Vanhatalo et al. (5), the only study that directly compares acute and prolonged supplementation (>1day),

their study suggests an additional effect of prolonged supplementation. Vanhatalo et al. (5) observed a

reduced O2 cost of exercise within 3 hours of the consumption of 5–6 mmol of NO3, but furthermore

observed that this effect can be preserved for at least 15 days provided that the same dose of nitrate is

consumed daily. Effects were observed throughout Vanhatalo et al.’s (5) study, with primary key outcome

measurements at 2.5 hours, 5 days and 15 days after supplementation. Even though Vanhatalo et al. (5)

observed improved exercise efficiency measured as a significant reduction in VO2submax, which continued

throughout the study, their results still favour 15 days of supplementation where greater and additional

positive effects in tems of oxygen uptake at the gas exchange threshold and peak power in contrast to only

an effect on VO2submax after 2.5 hours and 5 days. These effects were observed in connection with the

largest period of NO2 increase. However, this increase dropped from 59% to 46% from day 12 to 15, which

may indicate that longer supplementation has an upper limit and that tolerance to the supplementation

develops.

In dose perspective studies, after ingesting relative quantities of 5-8 mmol NO3, elevations typically ranged

from 50-150%, as shown in Figure 11. As described in three of the four studies, the largest NO2 changes

produced the biggest NO2 responses. The largest response was found by Wylie et al. (9) who prescribed 29

mmol NO3 to subjects over a period of 36 hours, resulting in an increase in NO2 of nearly 400%. Likewise, in

two of the other three studies, subjects ingested a larger quantity of NO3 than 8.4 mmol, which suggests

that higher NO3 doses resulted in higher plasma NO2 and, as stated above, this could theoretically increase

performance more. Supporting this, Wylie et al. (4) observed a larger ergogenic effect when supplementing

with 16.8 mmol NO3 as opposed to 4.2 mmol NO3. This shows a direct dose response relationship in

relation to the performance measurement and furthermore correlates the change in plasma NO2

concentrations from baseline to post ingestion to a decrease in end exercise VO2 (R=0.47; P<0.05).

However, even though Wylie et al. (4) observed a correlation and a direct measurement the exercise

protocol in terms of time to exhaustion did not increase further after consumption of 8.4 mmol NO3

compared with 16.8 mmol NO3, suggesting an upper limit of effect for an NO2 increase. Wylie et al. (9)

likewise presented a wide inter-individual variability which was observed where individuals’ plasma NO2

response to the ingestion of 16.8 mmol NO3 peak concentrations ranged from 493 to 1,523 nM, and time to

peak ranged from 130 to 367 min. Wylie et al. (9) also found that some of their subjects did not respond at


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4.2 mmol NO3. They were termed non-responders and they did respond at the higher 16.8 mmol NO3

supplementation. Several of the other included studies likewise speculated on the possibility of individual

response difference and proposed a responder versus non-responder relationship. This hypothesis has

especially been studied in trained subjects. For example, Wilkerson et al. (20) analysed their results by

dividing their study into responders (>30% increase in plasma NO2 concentration) and non-responders

(<30% increase in plasma NO2 concentration). This yielded data where the subjects who were categorized

as responders all improved their performance whereas the non-responders did not improve their

performance. The responders not only performed better, they also had a higher mean power output during

the 50-mile time trial and an increased power/VO2 ratio, contrary to the non-responders. These results are

supported by Christensen et al. (15) who, overall, found no significant effect in their ten elite cyclists.

However, in two of these subjects improvements of 2.5% and 8% were observed in their respective

VO2submax, especially in the time trial. The summarizing data therefore yield results of a responder/non-

responder relationship.

6.3.3. Summary

Independent substances seem to induce NO2 responses, which can eventually be transferred into an

ergogenic effect. However, due to the relatively low number of dependent substances studied it cannot be

finally concluded that these substances are more or less efficient than independent related supplements.

Furthermore, the existing literature presents a relatively similar number of studies finding ergogenic results

of NO-related supplementation in both acute (≤1 day) and prolonged supplementation (>1 day). However,

data on prolonged studies lasting longer than 6 days are limited. The only study which directly compares

supplementation duration longer than this, found greater and additional ergogenic effects with 15 days of

supplementation. Although it may be speculated that up to 15 days of supplementation may be preferred

for optimal performance enhancement, further studies are needed to determine and validate these

findings. In terms of dose, this review suggests a clear dose/response relationship. While the doses ranging

from 5-6 mmol NO3 in most individuals have been shown to enhance NO2, larger doses have been seen to

impose a greater impact on NO2 response. However, further studies need to be conducted in these fields to

determine if larger doses have a greater ergogenic effect. Furthermore, the literature presents evidence of

an individual response indicating that some individuals may benefit more from supplementation than

others, and some may need larger or smaller quantities to induce an effect.


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6.4. Limitations

This systematic review was conducted by one writer, which makes it vulnerable to selective bias. A way to

improve this would obviously be to include another writer, or simply to include more researches focusing

on narrower parts of the review e.g. adjusting the inclusion exclusion criteria. Furthermore, the study

involved searches of two of the most relevant databases for articles, but we have no way of knowing if this

limited the results by missing any other NO-related studies, thus affecting knowledge on the subject. It

could make a difference to the conclusions if missing studies differed significantly from those identified.

Likewise, additional studies could contribute stronger evidence supporting the conclusions drawn from the

review. In addition, attention should be paid to what The Cochrane Collaboration (60) describes as positive

bias, which proposes that publications with positive results are easier to identify, which may be the case

with this review since the majority of studies indicate positive results.

Even though the studies yield homogeneous results, in terms of study quality there seems to be great

heterogeneity in study design concerning subjects, exercise protocol and supplementation strategy across

the included studies, restricting the comparison between them. In this connection the majority of studies

have small sample sizes, as shown in Tables 3 and 4. This means that the statistical power is low, and a

bigger sample size would minimize the amount of sampling errors inherent in test results. However, since

the quality of the included studies is relatively high, this most likely minimized these errors, and given that

significant effects are harder to detect in smaller samples the results of these articles appear valid. This

could however have been determined if a meta-analysis was conducted, but this was not the purpose of

this extended paper.

7. Conclusion

This review combining the results of 23 studies indicates promising effects of NO-related supplementation,

with 12 studies presenting ergogenic results. Of the remaining studies, seven did not find any significant

effects, another three found both ergogenic and anti-ergogenic changes in performance-related

parameters, but did not find any decrease in final performance. Only one study found a direct decrease in

performance after supplementation.

The ergogenic effects of NO-related supplementation seem to decrease at higher fitness levels. The

included studies indicate an upper level at >60 mL O2/kg/min. In both low and high intensity exercise

ergogenic improvements were seen after supplementation. However, it seems that exercise performed at

higher intensities targeting local acidosis and hypoxia may be more responsive, which has been presented


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in a practical manner as improvements at the end of a race or the last couple of interval sessions. Acute (≤1

day) and prolonged (>1 day) supplementation both yielded positive performance-enhancing results.

However, a few comparative studies suggested that a prolonged strategy (>6 days) and higher NO3 dose

(≈8mmol NO3/dose) induce greater effects, which could be of significant interest taking the wide inter-

individual variability in the response to NO-related supplementation into consideration.

Since only a few studies report negative effects in terms of a lower VO2peak, but without any performance

decline, NO-related supplementation is a potentially relatively risk-free beneficial aid for aerobic exercise

performance.

8. Recommendations for future areas of research

The included studies ranged widely regarding subject characteristics, dietary intervention and the exercise

intervention performed, thereby producing controversial and inconclusive results. Ultimately, more and

larger studies are needed to determine if NO-related supplementation is truly effective, in which cases

supplementation would be beneficial and to further validate the findings. Further research could include

areas such as:

Training status: This review observes an upper level for ergogenic effect when supplementing with

NO-related substances. However, no specific studies have compared trained versus untrained

subjects, which could clarify the above proposed training status effect. Likewise, only two studies

examined elite subjects. Further studies of this population would be of great interest as this could

not only quantify a performance-enhancing effect but also more precisely determine the

underlying effects of NO-related supplementation.

Exercise measurements: The majority of studies targeted submaximal exercise protocols but only a

few of these examined longer trials (>30 minutes). Only shorter and intermittent trials (<30

minutes) revealed enhanced performance in terms of direct performance measurements. Further

research should therefore directly compare short and high intensity work with longer lower

intensity trials to elicit differences or similarities.

Supplementation strategy: Two studies compared different supplementation strategies in relation

to dose and timing. The dose-response curve is, as yet, relatively unknown which could clarify the

optimal way to implement supplementation. Further research is therefore needed to establish the

optimum practice for supplementation although the supplementation strategy should be suited to

the individual subject and fine-tuned during daily training to meet specific needs and produce

optimal response.


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