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Review Effect of dietary nitrate supplementation on metabolic rate during rest and exercise in human: A systematic review and a meta-analysis Mehdi Pawlak-Chaouch a , Julien Boissi ere a , François X. Gamelin a , Gr egory Cuvelier b , Serge Berthoin a , Julien Aucouturier a, * a Universite Droit et Sante Lille 2, URePSSS, EA7369, Equipe «Activite Physique, Muscle, Sante», Faculte des Sciences du Sport et de l'Education Physique, Ronchin, France b P^ ole Hainuyer, Laboratoire de l'Effort et du Mouvement, Haute Ecole Provinciale de Hainaut-Condorcet, Tournai, Belgium article info Article history: Received 28 September 2015 Received in revised form 27 November 2015 Accepted 5 January 2016 Available online 7 January 2016 Keywords: Dietary nitrate Metabolic rate Rest Exercise Chronic diseases abstract Background: Recent randomized controlled trials have suggested that dietary nitrate (NO 3 - ), found in beetroot and other vegetables, and inorganic NO 3 salts decrease metabolic rate under resting and ex- ercise conditions. Objective: Our aim was therefore to determine from a systematic review and meta-analysis whether dietary NO 3 supplementation signicantly reduces metabolic rate, expressed as oxygen uptake (VO 2 ), under resting and exercise conditions in healthy humans and those with cardiorespiratory diseases. Design: A systematic article search was performed on electronic databases (PubMed, Scopus and Web of Science) from February to March 2015. The inclusion criteria included 1) randomized controlled trials; 2) studies reporting the effect of NO 3 on VO 2 under resting and/or exercise conditions; 3) comparison between dietary NO 3 supplementation and placebo. Random-effects models were used to calculate the pooled effect size. Results: Twenty nine randomized placebo-controlled trials were included in the systematic review, and 26 of which were included in the meta-analysis. Dietary NO 3 supplementation signicantly decreases VO 2 during submaximal intensity exercise [0.26 (95% IC: 0.38, 0.15), p < 0.01], but not in the sub- analysis of subjects with chronic diseases [0.09 (95% IC: 0.50, 0.32), p ¼ 0.67]. When data were separately analyzed by submaximal intensity domains, NO 3 supplementation reduces VO 2 during moderate [0.29 (95% IC: 0.48,0.10), p < 0.01] and heavy [0.33 (95% IC: 0.54,0.12), p < 0.01] intensity exercise. When the studies with the largest effects were excluded from the meta-analysis, there is a trend for a VO 2 decrease under resting condition in dietary NO 3 supplementation [0.28 (95% IC: 0.62, 0.05), p ¼ 0.10]. Conclusion: Dietary NO 3 supplementation decreases VO 2 during exercise performed in the moderate and heavy intensity domains in healthy subjects. The present meta-analysis did not show any signicant effect of dietary NO 3 supplementation on metabolic rate in subjects with chronic diseases, despite enhanced exercise tolerance. © 2016 Elsevier Inc. All rights reserved. Abbreviations: I 2 , inconsistency value; NO 3 , nitrate; NO, nitric oxide; NOS, nitric oxide synthase; NO 2 , nitrite; PICOS, Participants, Interventions, Comparators, Outcomes, Study design; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses; t 2 , between-studies variance; VO 2 , oxygen uptake; VO 2max , maximal oxygen uptake. * Corresponding author. URePSSS, EA7369, Equipe «Activite Physique, Muscle, Sante», Faculte des Sciences du Sport et de l'Education Physique, Universite Lille 2, 9, Rue de l'Universite, 59790 Ronchin, France. E-mail address: [email protected] (J. Aucouturier). Contents lists available at ScienceDirect Nitric Oxide journal homepage: www.elsevier.com/locate/yniox http://dx.doi.org/10.1016/j.niox.2016.01.001 1089-8603/© 2016 Elsevier Inc. All rights reserved. Nitric Oxide 53 (2016) 65e76

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  • lable at ScienceDirect

    Nitric Oxide 53 (2016) 65e76

    Contents lists avai

    Nitric Oxide

    journal homepage: www.elsevier .com/locate/yniox

    Review

    Effect of dietary nitrate supplementation on metabolic rate during restand exercise in human: A systematic review and a meta-analysis

    Mehdi Pawlak-Chaouch a, Julien Boissi�ere a, François X. Gamelin a, Gr�egory Cuvelier b,Serge Berthoin a, Julien Aucouturier a, *

    a Universit�e Droit et Sant�e Lille 2, URePSSS, EA7369, Equipe «Activit�e Physique, Muscle, Sant�e», Facult�e des Sciences du Sport et de l'Education Physique,Ronchin, Franceb Pôle Hainuyer, Laboratoire de l'Effort et du Mouvement, Haute Ecole Provinciale de Hainaut-Condorcet, Tournai, Belgium

    a r t i c l e i n f o

    Article history:Received 28 September 2015Received in revised form27 November 2015Accepted 5 January 2016Available online 7 January 2016

    Keywords:Dietary nitrateMetabolic rateRestExerciseChronic diseases

    Abbreviations: I2, inconsistency value; NO3�, nitrateoxide synthase; NO2�, nitrite; PICOS, Participants,Outcomes, Study design; PRISMA, Preferred Reportingand Meta-Analyses; t2, between-studies variance; Vmaximal oxygen uptake.* Corresponding author. URePSSS, EA7369, Equipe

    Sant�e», Facult�e des Sciences du Sport et de l'Educatio9, Rue de l'Universit�e, 59790 Ronchin, France.

    E-mail address: [email protected] (J

    http://dx.doi.org/10.1016/j.niox.2016.01.0011089-8603/© 2016 Elsevier Inc. All rights reserved.

    a b s t r a c t

    Background: Recent randomized controlled trials have suggested that dietary nitrate (NO3- ), found in

    beetroot and other vegetables, and inorganic NO3� salts decrease metabolic rate under resting and ex-ercise conditions.Objective: Our aim was therefore to determine from a systematic review and meta-analysis whetherdietary NO3

    � supplementation significantly reduces metabolic rate, expressed as oxygen uptake (VO2),under resting and exercise conditions in healthy humans and those with cardiorespiratory diseases.Design: A systematic article search was performed on electronic databases (PubMed, Scopus and Web ofScience) from February to March 2015. The inclusion criteria included 1) randomized controlled trials; 2)studies reporting the effect of NO3� on VO2 under resting and/or exercise conditions; 3) comparisonbetween dietary NO3� supplementation and placebo. Random-effects models were used to calculate thepooled effect size.Results: Twenty nine randomized placebo-controlled trials were included in the systematic review, and26 of which were included in the meta-analysis. Dietary NO3� supplementation significantly decreasesVO2 during submaximal intensity exercise [�0.26 (95% IC: �0.38, �0.15), p < 0.01], but not in the sub-analysis of subjects with chronic diseases [�0.09 (95% IC: �0.50, 0.32), p ¼ 0.67]. When data wereseparately analyzed by submaximal intensity domains, NO3� supplementation reduces VO2 duringmoderate [�0.29 (95% IC: �0.48,�0.10), p < 0.01] and heavy [�0.33 (95% IC: �0.54,�0.12), p < 0.01]intensity exercise. When the studies with the largest effects were excluded from the meta-analysis, thereis a trend for a VO2 decrease under resting condition in dietary NO3� supplementation [�0.28 (95%IC: �0.62, 0.05), p ¼ 0.10].Conclusion: Dietary NO3

    � supplementation decreases VO2 during exercise performed in the moderate andheavy intensity domains in healthy subjects. The present meta-analysis did not show any significanteffect of dietary NO3� supplementation on metabolic rate in subjects with chronic diseases, despiteenhanced exercise tolerance.

    © 2016 Elsevier Inc. All rights reserved.

    ; NO, nitric oxide; NOS, nitricInterventions, Comparators,Items for Systematic ReviewsO2, oxygen uptake; VO2max,

    «Activit�e Physique, Muscle,n Physique, Universit�e Lille 2,

    . Aucouturier).

    Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:[email protected]://crossmark.crossref.org/dialog/?doi=10.1016/j.niox.2016.01.001&domain=pdfwww.sciencedirect.com/science/journal/10898603http://www.elsevier.com/locate/ynioxhttp://dx.doi.org/10.1016/j.niox.2016.01.001http://dx.doi.org/10.1016/j.niox.2016.01.001http://dx.doi.org/10.1016/j.niox.2016.01.001

  • M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e7666

    Contents

    1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

    2.1. Type of studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662.2. Type of interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672.3. Type of outcome measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672.4. Search strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672.5. Study selection and data extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 672.6. Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

    3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683.1. Study selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683.2. Study characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683.3. Risk of bias within studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693.4. Results of individual studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693.5. Synthesis of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

    3.5.1. Pooled effect size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693.5.2. Subgroup analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703.5.3. Influence of studies with the largest effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.5.4. Publication bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.5.5. Heterogeneity and inconsistency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

    4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734.1. Summary of evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734.2. Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

    5. Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

    1. Introduction Larsen et al. (2014) also showed in a randomized controlled

    Lowoxygen availability limits the sustainablemetabolic rate in awide range of conditions, such as in chronic cardiorespiratory dis-eases, environmental hypoxia or in athletes when oxygen carryingcapacity reaches its maximal during intense exercise [1e3]. Meta-bolic rate is minimal under resting, thermoneutral, and post-absorptive conditions, comprising the energy expenditure requiredto sustain vital body functions [4]. Brain, liver, heart, and kidneysaccount for 60e70% of resting energy expenditure, whereas skel-etal muscles account for 20e30% in humans [5]. During exercise,metabolic rate can be increased up to 20-fold during maximal in-tensity exercise in athletes to meet to the energy needs of workingmuscles [4]. Metabolic rate is commonly expressed as the rate ofoxygen uptake (VO2), which reflects whole body oxidative meta-bolism and is measured by indirect calorimetry [6]. There is how-ever little possibility to reduce resting metabolic rate, or metabolicrate for a givenwork rate during exercise [7]. Nitric oxide (NO) is anubiquitous signaling molecule produced through the NO synthasepathway that possesses the ability to lower metabolic rate throughan increase in mitochondrial efficiency caused by factors such asthe inhibition of enzyme cytochrome c oxidase activity by NO, orthe inhibition of uncoupled respiration [8]. NO synthesis throughthe nitric oxide synthase (NOS) pathway is however dependent onL-arginine and oxygen availability, and becomes limited in hypoxia[9]. However, dietary nitrate (NO3- ) found in abundance in greenleafy and root vegetables have been shown to represent animportant alternative source of NO [10]. Dietary NO3� are absorbedfrom the small intestine, and after an entero-salivary recirculationand concentration into the saliva, are converted into nitrite (NO2- )by oral NO3� reductase bacteria [10]. The swallowed salivary NO2�

    are either reduced to NO and other nitrogen species in the acidicstomach or absorbed from the intestine [10].

    In humans, increased NO bioavailability was first shown toresult in decreased VO2 during exercise [7,11,12]. Recently,

    trial that resting metabolic rate is reduced by 4.2% in healthysubjects following a 3-day dietary NO3� supplementation [13].The decrease in resting metabolic rate with dietary NO3� sup-plementation was proposed to mimic the beneficial effect ofcalorie restriction or resveratrol supplementation, whichwere previously shown to improve metabolic health parameters[14,15]. A number of studies have also been conducted inpatients with chronic disease conditions that severely impairoxygen delivery and/or utilization, such as chronic obstructivepulmonary disease, heart failure, or peripheral arterial disease[16e20].

    Our purpose was therefore to conduct a systematic review andmeta-analysis of the randomized controlled trials exploring theeffect of inorganic NO3� supplementation on the metabolic rate,expressed as VO2, under resting and/or exercise conditions. A sec-ondary purpose of the meta-analytical procedures was to deter-mine factors, such as population characteristics, supplementationduration, and dietary NO3� dose that could be responsible of thehypothesized changes in metabolic rate.

    2. Methods

    The systematic review and the meta-analysis were conductedaccording to the established guidelines in Cochrane Handbook forSystematic Reviews of Interventions, and were reported accordingto the PRISMA (Preferred Reporting Items for Systematic Reviewsand Meta-Analyses) guidelines [21]. The criteria of PICOS (Partici-pants, Interventions, Comparators, Outcomes, Study design)approach are presented in Table 1.

    2.1. Type of studies

    Randomized clinical trials that investigated the effect of dietaryNO3� under the form of inorganic NO3� salts or present in beetroot

  • Table 1Description of PICOS approach used in the systematic review and meta-analysis.

    Components Criteria

    Participants Adult >18 years with or without chronic disease conditions that severely impair oxygen delivery and/or utilization in particular.Interventions Increased dietary NO3� intake by consumption of vegetables rich in NO3� such as beetroot, or nitrate salt solutions without any restriction regarding

    supplementation characteristics.Comparators Placebo with negligible nitrate contentOutcomes Metabolic rate expressed as VO2Study design Randomized controlled trials

    Crossover designParallel designDouble- or single-blind, or otherwise

    NO3�: nitrate; VO2: oxygen uptake.

    Table 2Criteria for the determination of exercise intensity domains.

    Intensity domains Physiological demarcation Corresponding % VO2max or aerobic peak power

    Moderate Below the gas exchange threshold 80%Maximal Exercise performed up to subject's exhaustion with the attainment

    of VO2max or VO2peak

    The critical power is defined as the intensity above which the attainment of VO2max is elicited, which is maintained up to exhaustion.

    M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e76 67

    juice on metabolic rate, expressed as VO2, in humanwere included.The following experimental variables were extracted from eachstudy: resting or exercise conditions, supplementation duration,dosage, adherence to protocols, dropout rate, duration of thewashout period between dietary NO3� and placebo supplementa-tions, and adverse events. Participant characteristics were alsoextracted with the following information whenever available: age,number of male and female subjects, health status (healthy, chronicobstructive pulmonary disease, heart failure, and peripheral arte-rial disease), anthropometric characteristics, physical activity orexercise training level and maximal oxygen uptake (VO2max) toreflect aerobic fitness.

    2.2. Type of interventions

    The studies included achieved increased dietary NO3� intake byconsumption of beetroot juice rich in inorganic NO3� or nitrate saltsdissolved in aqueous solutions (potassium nitrate or sodium ni-trate). Supplementation duration, dietary NO3� dose, type of sup-plementation (beetroot juice or salts) were parameters used aspotential sources of heterogeneity in random-effects models. Forstudies where metabolic rate was assessed during exercise, wedefined the exercise intensity domain asmoderate, heavy, or severebased on the existing literature [22,23] in order to determinewhether the effect of dietary NO3� supplementation is affected byexercise intensity. Studies where exercise was performed in themoderate, heavy and severe intensity domains were also pooledtogether for a separate analysis of metabolic rate during submaxi-mal intensity exercise (

  • 3314 records were identified through database searching

    2654 records were removed for duplicate

    660 records were screened by titles

    57 records were screened by abstracts

    9 full-text articles were excluded for:

    - Oxygen uptake was not measured (7)

    - No placebo group (2)

    38 full-text articles were assessed for eligibility

    29 studies were included in qualitative synthesis

    26 studies were included in quantitative synthesis

    (meta-analysis)

    3 additional records were identified through other sources

    noitacifitnedIgnineercS

    ytilibigilEdedulcnI

    Fig. 1. Flow chart of the selection process in literature search.

    M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e7668

    effect size for VO2 under resting and exercise conditions. Data arereported as standardized mean differences for VO2 at rest andduring exercise with 95% Confidence Interval (CI), and presented inforest plots. The threshold for statistical significance was set atp ¼ 0.05. Heterogeneity among studies was evaluated using theCochrane's Q test (p < 0.10) and I2 values (low � 25%,moderate � 50%, high � 75%), and between-studies variance wascalculated (Tau2, t2 > 1). Subgroup analyses were performed todetermine the effect of the following factors on random-effectsmodels: supplementation form (inorganic NO3� salts or beetrootjuice), experimental condition (normoxia or hypoxia), dietary NO3�

    dose, supplementation duration, plasma NO3�/NO2- response (highor low NO3� and/or NO2� response) and health status (healthy orpathological subjects). The median value for each parameterincluded in subgroup analyses was used to separate into two sub-groups: dietary NO3� dose (high > 7.5 mmol vs. low �7.5 mmol),supplementation duration (short� 3 days vs. long >3 days), andchange in plasma NO3�/NO2- levels (high NO3� > þ574% vs. lowNO3� � þ574%; high NO2� > þ115.5% vs. low NO2� � þ115.5%).Sensitivity analysis was performed to determine the influence ofthe studies with the largest effects on the pooled effect size ofresting studies. The risk assessment of bias was performed sepa-rately by two authors (M.P.-C. and J.A.) in accordancewith CochraneRisk of Bias Tool's items. Identification of publication bias wasperformed by funnel plot analysis [24].

    3. Results

    3.1. Study selection

    Based on selected search terms, the initial search in electronic

    database retrieved 3314 articles, of which 53 articles were poten-tially eligible for inclusion after title and abstract reading. There-after, 29 articles were identified as eligible, and included in thesystematic review for qualitative analysis. Twenty-six articles wereincluded in meta-analysis to perform quantitative analysis. Theremaining studies were excluded because: data were unavailableafter request to the corresponding author, a heterogeneity in workrate between the NO3� and placebo conditions in exercise studies,and no assessment of the change in NO3� and NO2� plasma levels. Aflow chart of the literature search procedure is presented in Fig. 1.

    3.2. Study characteristics

    Most included studies were randomized, double- or single-blind, placebo controlled, parallel or crossover trials, and con-ducted between 2007 and 2015. Twenty-three studies were con-ducted in active or trained, young and healthy subjects. One studywas conducted in the elderly (63.5 ± 3.0 years) [25]. One study wasconducted in overweight, but otherwise healthy subjects [26]. Fourstudies were performed in populations with chronic disease con-ditions. Two studies were conducted in patients with chronicobstructive pulmonary disease [16,19], one study in patients withperipheral arterial disease [17], and one study in patients withheart failure with preserved ejection fraction [20]. The meta-analysis included a total of 264 participants with 7e17 partici-pants per study. The main characteristics of the 29 studies includedare presented in Supplemental Table 1.

    NO3� supplementation was achieved by intake of inorganic NO3�

    salts (nitrate sodium or nitrate potassium solutions) in 6 studiesand beetroot juice in 23 studies. The following placebo were used:equimolar sodium chloride solutions in 5 studies, isovolumetric

  • M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e76 69

    NO3�-free maltodextrin solution in 1 study, NO3�-depleted beetrootjuice in 12 studies, apple-blackcurrant juice in 4 studies, black-currant juice in 3 studies, orange juice in 2 studies, prune juice in 1study and tomato juice in 1 study. There was a single ingestion ofdietary NO3� in 13 studies and a supplementation duration rangedfrom 3 to 15 days in 15 studies with washout period between 2 and14 days, with the exception of one study with parallel design,where the placebo and beetroot juice supplementation were initi-ated 4 days before baseline testing and continued during 6weeks ofexercise training [27]. The last ingestion most often occurred2.5e3 h before VO2 measurement, which corresponds to peakplasma NO2� concentrations after dietary NO3� ingestion [28,29],with the exception of one study where the ingestionwas 1 h beforeassessment [13]. The daily amount of NO3� ingested ranged from 5.1to 19.5 mmol.

    Regarding additional dietary NO3� intake, participants wereasked to refrain from consuming NO3�-rich food items in 12 studies,three of which applied nutritional guidelines [30] or standardizeddinner and breakfast [27,31]. Seventeen studies reported no dietaryrestriction, but 10 of these studies indicated that the participantswere instructed to replicate their food intake in the two conditions(nitrate vs placebo), and 4 studies used standardized meals beforeexperimental tests following supplementation periods [32e35].The subjects were instructed to refrain from caffeine and alcohol 6and 24 h before tests in studies investigating the effect of supple-mentation on exercise performance. The study participants wereasked also to restrain from strenuous activity within the 24e48 hpreceding testing sessions. In all studies, the subjects wereinstructed not to use antibacterial mouthwashes that inhibit theNO3� to NO2� conversion by oral anaerobic bacteria [28].

    3.3. Risk of bias within studies

    Most studies includedwere considered as having low risk of biasaccording to Cochrane Risk of Bias Tool's items [36]. Two studieswere rated as having high risk of bias because of the lack of blindingfor experimental beverages of participants and research personnel.A number of studies were rated as having an unclear risk of bias inone or more items as follows: random sequence generation in 1study, blinding of participants and personnel in 9 studies, blindingof experimental outcome assessments in 3 studies, and incompleteoutcome data in 3 studies. The allocation of interventions wasexclusively randomized, andwashout period and dropout ratewerereported in all trials. The risk of bias assessment is reported inSupplemental Fig. 1. Eligible trials were mostly double- (n ¼ 16) orsingle-blind (n ¼ 9) when placebo with different taste, smell andappearance compared to dietary NO3� supplementation were used.Seven studies reported that the participants were concealed toexperimental hypotheses, whereas the purpose of trials wascommunicated as the comparison of physiologic responses aftertwo beverage intakes or treatment solutions. Otherwise, the au-thors notified that the study participants were not informed aboutthe potential physiologic effects of inorganic NO3� salts and beetrootjuice supplementation. Eighteen studies did not report whether thestudy participants were aware of the properties of inorganic NO3�

    and of the true aim of study. In two trials, the study participantswere informed that the purpose of the studywas to test the effect ofa NO3�-rich beverage on exercise performance [16,33]. Eighteenstudies reported funding sources and 8 studies disclosed potentialconflict of interest.

    3.4. Results of individual studies

    Dietary NO3� supplementation was well tolerated, and no sideeffects were reported. Some participants reported beeturia (red

    urine) and red stools after beetroot juice supplementation, butthese events were considered benign.

    The summary of main results in each included study is displayedin Supplemental Table 1. Two studies reported a reduced VO2under resting condition after dietary NO3� supplementation [13,31],one of which was performed under hypoxic condition [31]. Onlyone study was designed to specifically investigate the effect of di-etary NO3� on resting metabolic rate and reported a 4.2% decrease[13]. Four studies did not report any effect of dietary NO3� supple-mentation on baseline VO2 measurements, which were performedbefore the beginning of exercise tolerance tests [12,26,30,37].VO2 was significantly decreased following dietary NO3� supple-mentation in 12 studies during moderate intensity steady-stateexercise [7,11,12,26,31,32,35,38e40], heavy intensity exercise[7,26,32,41,42], and severe intensity exercise [7,12,26,38]. Thedecreased VO2 was associated with enhanced exercise toleranceduring severe intensity exercise [11,12,35,38]. Six studies reportedno change in VO2 during exercise with dietary NO3� supplementa-tion [25,33,34,37,43,44]. Several studies investigated the effect ofdietary NO3� on time trial performance and reported VO2 mea-surements. One study showed that VO2 was significantly decreasedduring a 4-min all-out maximal effort [45], but 2 studies did notreport any effect of dietary NO3� supplementation on VO2 during 4-km and 16.1-km time trials and during 50 mile time trials,respectively [46,47]. However, the power output to VO2 ratio duringexercise significantly increased in the two latter studies, indicativeof a decrease in the O2 cost of exercise. Two studies showed thatVO2max was decreased during maximal intensity exercise [30,48]without change in time to exhaustion after inorganic NO3� sup-plementation, and one study reported no effects on VO2max [7].Finally, one study investigated the combined effect of beetroot juicesupplementation and hypoxic endurance training on aerobic fitnessand reported a similar increase in VO2max between NO3� supple-mentation and placebo [27].

    Regarding patients with chronic disease conditions, 2 studies inchronic obstructive pulmonary disease did not report any change inVO2 after beetroot juice supplementation [16,19], despite increasedexercise capacity [16]. In patients with peripheral arterial disease,exercise tolerance was enhanced and the onset of claudication painwas delayed, but VO2 was significantly reduced at the first stage ofthe incremental exercise test only [17]. In patients with heart failurewith preserved ejection fraction, beetroot juice supplementationresulted in higher VO2peak and increased total work achieved dur-ing a supine-cycle maximal exercise test [20]. Regarding metabolicrate under non-exercise conditions, baseline VO2 measured beforeexercise tolerance tests was unchanged following beetroot juiceingestion in 2 studies in patients with chronic obstructive pulmo-nary disease [16,19]. However, a study conducted in patients withperipheral arterial disease showed a trend toward a lower restingVO2 after beetroot juice ingestion [17].

    3.5. Synthesis of results

    3.5.1. Pooled effect sizeDietary NO3� supplementation resulted in a significant VO2

    decrease during submaximal intensity exercise [�0.26 (95%IC: �0.38, �0.15), p < 0.01] (Fig. 2). When data from the threesubmaximal intensity domains were analyzed separately, dietaryNO3� supplementation resulted in significant VO2 decrease duringmoderate intensity exercise [�0.29 (95% IC:�0.48,�0.10), p < 0.01](Fig. 3) and heavy intensity exercise [�0.33 (95% IC: �0.54,�0.12),p < 0.01] (Fig. 4). However, there was no significant effect of dietaryNO3� relative to placebo under resting condition [0.01 (95%IC: �0.47, 0.50), p ¼ 0.96] (Fig. 5), during severe intensity exercise[�0.14 (95% IC: �0.38, 0.09), p ¼ 0.24] (Fig. 6) and during maximal

  • Fig. 2. Forest plot of the effect size for the change in VO2 during submaximal intensity exercise with dietary NO3� supplementation.

    M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e7670

    intensity exercise [0.02 (95% IC: �0.42, 0.46), p ¼ 0.93] (Fig. 7).

    3.5.2. Subgroup analysesSubgroup analyses showed no effect of any of the selected pa-

    rameters on VO2 during submaximal intensity exercise, except fromhealth status, where dietary NO3� supplementation significantlydecreased VO2 in healthy subjects [�0.28 (95% IC: �0.40, �0.16),p < 0.01], but not in patients with chronic diseases [�0.09 (95%IC: �0.50, 0.32), p ¼ 0.67].

    Subgroup analysis for each exercise intensity domain showed

    that treatment form did not change the effect of NO3� supplemen-tation on VO2, with significant VO2 decrease during moderate[beetroot juice: �0.27 (95% IC: �0.50, �0.04), p ¼ 0.02; inorganicNO3� salts: �0.38 (95% IC: �0.76, 0.00), p ¼ 0.05] and heavy in-tensity exercise [beetroot juice: �0.30 (95% IC: �0.55, �0.04),p ¼ 0.02; inorganic NO3� salts: �0.41 (95% IC: �0.81, �0.01),p ¼ 0.04].

    Subgroup analysis by dose showed that low NO3� dose had asignificant effect during moderate intensity exercise [�0.41 (95%IC: �0.69, �0.12), p < 0.01], but not high NO3� dose [�0.17 (95%

  • Fig. 3. Forest plot of the effect size for the change in VO2 during moderate intensity exercise with dietary NO3� supplementation.

    Fig. 4. Forest plot of the effect size for the change in VO2 during heavy intensity exercise with dietary NO3� supplementation.

    Fig. 5. Forest plot of the effect size for the change in VO2 under resting condition with dietary NO3� supplementation.

    M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e76 71

  • Fig. 6. Forest plot of the effect size for the change in VO2 during severe intensity exercise with dietary NO3� supplementation.

    Fig. 7. Forest plot of the effect size for the change in VO2 during maximal intensity exercise with dietary NO3� supplementation.

    M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e7672

    IC: �0.43, 0.09), p ¼ 0.20]. High NO3� dose significantly decreasesVO2 during heavy intensity exercise [�0.53 (95% IC: �1.06, �0.01),p ¼ 0.05], and low NO3� dose was associated with a trend towardsignificant effect size [�0.24 (95% IC: �0.50, 0.02), p ¼ 0.07].

    The supplementation duration affects the change in VO2, withthe longer supplementation duration resulting in a significant VO2decrease during moderate intensity exercise [�0.62 (95%IC:�1.05,�0.19), p < 0.01], that is not observed with short durationsupplementation [�0.14 (95% IC: �0.35, 0.07), p ¼ 0.19]. Surpris-ingly, short duration supplementation was associated with a sig-nificant decrease in VO2 during heavy intensity exercise [�0.30(95% IC: �0.57, �0.04), p ¼ 0.02], but not with long duration sup-plementation [�0.38 (95% IC: �0.87, 0.10), p ¼ 0.12].

    No change was found under resting condition and in severe andmaximal intensity exercise following the subgroup analyses bysupplementation form, dietary NO3� dose and supplementationduration.

    NO3� supplementation has no effect on VO2 in patients withchronic disease conditions [resting condition:�0.04 (95% IC:�0.57,0.48), p ¼ 0.87; moderate intensity exercise: 0.01 (95% IC: �0.50,0.51), p ¼ 0.98]. When the patients with chronic disease conditionswere excluded from the analysis, healthy subjects had a decreasedVO2 during moderate intensity exercise [�0.33 (95%IC: �0.54, �0.13), p < 0.01].

    Subgroup analysis for hypoxic condition is not reported becauseof the small number of studies performed under hypoxia [27,31,42].Subgroup analysis based on plasma NO3�/NO2- response is not re-ported because of the different analytical approaches used to assessNO3�/NO2- that results in extremely large differences of concentra-tions between studies.

    3.5.3. Influence of studies with the largest effectsWhen the exclusion of studies with the largest effects was

    performed, the random-effects models were affected under restingcondition with a trend toward a VO2 decrease [�0.28 (95%IC: �0.62, 0.05), p ¼ 0.10]. The sensitivity analysis also showed thatthe exclusion of studies with the largest effects did not change theeffects of NO3� supplementation on VO2 during submaximal in-tensity exercise [�0.23 (95% IC: �0.35, �0.11), p < 0.01], and whenthe three submaximal intensity domains were analyzed separately(moderate intensity exercise [�0.26 (95% IC: �0.43, �0.08),p < 0.01], heavy intensity exercise [�0.26 (95% IC: �0.48, �0.04),p¼ 0.02], and severe intensity exercise [�0.11 (95% IC: �0.35, 0.14),p ¼ 0.38]). The exclusion of studies with the largest effects did notchange the effect of NO3� supplementation during exercise per-formed at maximal intensity [�0.17 (95% IC: �0.47, 0.13), p ¼ 0.27].

    The results of the subgroup analyses for the factors potentiallyaffecting metabolic rate were unchanged for moderate and severeintensity exercise when the studies with the largest effects wereexcluded. When the analysis was limited to beetroot juice supple-mentation [�0.19 (95% IC: �0.46, 0.07), p ¼ 0.15] and high NO3�dose [�0.31 (95% IC: �0.74, 0.11), p ¼ 0.15], the effects on VO2 wasno longer significant during heavy intensity exercise. When theanalysis was limited to healthy subjects, there were trends towardssignificant VO2 decrease under resting condition [�0.37 (95%IC: �0.80, 0.05), p ¼ 0.08] and during maximal intensity exercise[�0.29 (95% IC: �0.63, 0.06), p ¼ 0.10].

    3.5.4. Publication biasThe analysis of funnel plots indicates an overall symmetric

    distribution of the studies around the pooled effect size for

  • M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e76 73

    moderate, heavy and severe intensities of exercise, suggesting nopublication bias. Under resting condition as well as for pooledsubmaximal intensity exercises andmaximal intensity exercise, thefunnel plots showed an overall asymmetric distribution of effectsizes of the individual studies (See Supplemental Fig. 2A, B and C)that may be related to the significant heterogeneity in thesemodels(see thereafter).

    3.5.5. Heterogeneity and inconsistencyWe observed no significant heterogeneity and inconsistency for

    submaximal intensity exercise [Q ¼ 45.12 (p ¼ 0.80), t2 ¼ 0,I2 ¼ 0%]. However, there was significant heterogeneity for longduration supplementation [Q ¼ 28.37 (p ¼ 0.10), t2 ¼ 0.10] and lowinconsistency [I2 ¼ 30%], which disappeared whenwe excluded thestudies with the largest effects [Q ¼ 15.57 (p ¼ 0.62), t2 ¼ 0,I2 ¼ 0%].

    There was high heterogeneity in random-effects models forresting condition [Q ¼ 39.77 (p < 0.01), t2 ¼ 0.51] and maximalintensity exercise [Q ¼ 17.99 (p ¼ 0.02), t2 ¼ 0.25], with high[I2 ¼ 72%] and moderate [I2 ¼ 56%] inconsistency, respectively.Under resting condition, beetroot juice supplementation [Q¼ 36.70(p < 0.01), t2¼1.08], lowNO3� dose [Q¼ 36.86 (p < 0.01), t2¼ 0.91],long duration supplementation [Q¼ 34.24 (p < 0.01), t2¼ 6.74] andhealthy subjects [Q ¼ 37.13 (p < 0.01), t2 ¼ 0.74] had large het-erogeneity, and high inconsistency [I2 ¼ 84%, 81%, 94%, and 78%,respectively]. Regarding maximal intensity exercise, the subgroupanalyses for beetroot juice supplementation [Q ¼ 15.03 (p ¼ 0.01),t2 ¼ 0.40], low NO3� dose [Q ¼ 16.82 (p ¼ 0.01), t2 ¼ 0.39], longduration supplementation [Q ¼ 14.83 (p < 0.01), t2 ¼ 0.87] andhealthy subjects [Q ¼ 17.09 (p ¼ 0.02), t2 ¼ 0.31] resulted inincreased heterogeneity and inconsistency [I2 ¼ 67%, 64%, 80%, and59%, respectively].

    The exclusion of the studies with the largest effects alteredsubstantially the random-effects models, with a reduced hetero-geneity and inconsistency under resting condition [Q ¼ 15.35(p ¼ 0.08), t2 ¼ 0.12, I2 ¼ 41%] and during maximal intensity ex-ercise [Q ¼ 5.13 (p ¼ 0.64), t2 ¼ 0, I2 ¼ 0%]. Regarding sources ofheterogeneity, the sensitivity analyses were associated withreduced inconsistency with beetroot juice supplementation[I2 ¼ 69%], low NO3� dose [I2 ¼ 57%] and healthy subjects only[I2 ¼ 48%] under resting condition. The heterogeneity remainedsignificant in the beetroot juice supplementation subgroup[Q ¼ 12.93 (p ¼ 0.01), t2 ¼ 0.36] and low NO3� dose subgroup[Q ¼ 11.50 (p ¼ 0.04), t2 ¼ 0.22]. The exclusion of the studies withthe largest effects in maximal intensity exercise model changedboth the heterogeneity and inconsistency as follows: beetroot juicesupplementation [Q ¼ 4.04 (p ¼ 0.40), t2 ¼ 0, I2 ¼ 1%], low NO3�dose [Q ¼ 2.94 (p ¼ 0.71), t2 ¼ 0, I2 ¼ 0%], long duration supple-mentation [Q ¼ 2.18 (p ¼ 0.34), t2 ¼ 0.02, I2 ¼ 8%] and healthysubjects [Q ¼ 2.95 (p ¼ 0.82), t2 ¼ 0, I2 ¼ 0%].

    4. Discussion

    4.1. Summary of evidence

    The main findings from the present meta-analysis are that di-etary NO3� supplementation is associated with i) a significantlydecreased metabolic rate when exercise is performed in the mod-erate and heavy intensity domains, and ii) that inorganic NO3� saltsand beetroot juice supplementation induced similar effect on VO2in these two exercise intensity domains. The results are howeverinconclusive regarding the effects of dietary NO3� dose and sup-plementation duration. However, there was no significant effect onVO2 in severe and maximal intensity exercise, suggesting that theeffects of NO on the O2 cost of exercise are alleviated as the

    intensity of exercise approaches the maximum. The exercise in-tensity domains used in the present study were based on thresh-olds expressed as percentage VO2peak that may be debatable. Themain effects of dietary NO3� supplementation on VO2 under restingand exercise are summarized and displayed in Table 3.

    In contrast to moderate intensity exercise, we found that dietaryNO3� supplementation does not significantly alter metabolic rateunder resting condition, although a trend toward a significantlyreduced metabolic rate appeared when the studies with the largesteffects were excluded. The lack of effects of NO3� supplementationunder resting condition may be related to physicochemical condi-tions in high metabolic rate organs and muscle that weaklycontribute to the activation of the nitrate-nitrite-NO pathway. Incontrast, NO2� bioactivity is enhanced in conditions of low oxygenpressure and pH by enhanced NO2� conversion into NO species [10].This nitrite reductase activity could thus be hypothesized to bestrongly activated during conditions such as exercise and/orchronic cardiac and respiratory disease. However, only one studyreported a trend towards a lower VO2 under resting condition inperipheral arterial disease [17], and results of the meta-analysissuggest that the effects of dietary nitrate supplementation on VO2is decreased as the intensity of exercise increases, which does notsupport the hypotheses. Otherwise, most eligible studies includedin the meta-analysis for metabolic rate under resting conditionprovided less reliable measurement of metabolic rate under restingcondition. Indeed, the primary purpose of these studies was toinvestigate the effect of dietary NO3� on the O2 cost of exercise insubjects who did not fast overnight, and VO2 was measured at restbefore the beginning of exercise tests, with indirect calorimetrydevices used for VO2 measurement during exercise, which may beless adequate for measurement under resting condition. Thusfurther studies are required to determine whether dietary NO3�

    supplementation significantly affects resting metabolic rate. Inaddition, only one studywas specifically designed to investigate theeffect of dietary NO3� on resting metabolic rate, and used the mostappropriate methodology with the VO2 measurement performedwith a ventilated hood and subjects tested after an overnight fast,and showed a significant decline in resting metabolic rate [13].Larsen et al. also showed a NO2� dose-dependent increase ofmitochondrial p50 for oxygen in vitro, which was also observedafter NO3� supplementation [39], and is strongly (R2 ¼ 0.66) andnegatively associated with basal metabolic rate [49]. Hence, anincrease in NO2� bioavailability through dietary NO3� supplemen-tation can reasonably be expected to result in decreased basalmetabolic rate. Only a limited number of treatments have previ-ously been reported to decrease resting metabolic rate. Forexample, basal metabolic rate was shown to decrease by 4% inhealthy obese subjects supplemented during 30 days with resver-atrol, a natural phenol present in various dietary vegetable com-ponents [15]. Resting metabolic rate has also been shown todecrease by 6% in healthy overweight subject who had followed a6-month caloric restriction [50,51].

    Regarding the effect of dietary NO3� in populations with car-diovascular and respiratory diseases, we did not observe a signifi-cant decrease in metabolic rate that could counteract the impairedoxygen carrying capacity. It is important to note that the studiesincluded or not in the meta-analysis reported enhanced exercisetolerance in patients with chronic obstructive pulmonary disease[16,18], peripheral arterial disease [17], and heart failure withpreserved ejection fraction [20]. In addition and in contrast to ourhypothesis, beetroot juice supplementation in patients with heartfailure with preserved ejection fraction resulted in increasedVO2peak, which was associated with enhanced vasodilatory reserveand cardiac output at peak exercise [20]. Of note, another studyreported an increase in VO2max after 15 days of beetroot juice

  • Table 3Summary of dietary nitrate supplementation effects on VO2.

    Random-effects models Effect size Significance (p < 0.05)

    Resting condition 0.01 (95% IC: �0.47, 0.50) p ¼ 0.96Submaximal intensity exercise �0.26 (95% IC: �0.38, �0.15) p < 0.01Moderate intensity exercise �0.29 (95% IC: �0.48, �0.10) p < 0.01Heavy intensity exercise �0.33 (95% IC: �0.54,�0.12) p < 0.01Severe intensity exercise �0.14 (95% IC: �0.38, 0.09) p ¼ 0.24Maximal intensity exercise 0.02 (95% IC: �0.42, 0.46) p ¼ 0.93

    M. Pawlak-Chaouch et al. / Nitric Oxide 53 (2016) 65e7674

    supplementation in healthy subjects, in which an increased localmuscle perfusion and cardiac output were hypothesized to befactors contributing to this change [40]. We were also unable todetermine the effect of either aerobic fitness level (VO2max) or ac-tivity/training status, as many studies included subjects with largerange of VO2max or did not report physical activity and traininghistory, which did not allow categorizing the populations accordingto the aerobic fitness and/or activity/training status. It was recentlyfound that the decrease in the O2 cost of exercise was negativelycorrelated with VO2max, and that the increase in NO2� plasma levelswith dietary NO3� supplementationwas lower in subjects with highVO2max [52]. These results confirm those of previous studies wherethe NO3� supplementation did not affect VO2, exercise tolerance orexercise performances in highly endurance trained subjects[33,34,43,47].

    4.2. Limitations

    There are limitations inherent to meta-analysis that should beconsidered in the interpretation of the results due the smallnumber of studies in several models and the reduced sample size inmost studies. A significant heterogeneity also appeared underresting condition and duringmaximal intensity exercise, whichwasreduced when the studies with the largest effects were excluded insensitivity analysis.

    The meta-analysis included a narrow range of subjects in termsof age, gender, health status and physical activity level. The over-representation of active or highly endurance trained, young, andhealthymale participants is a limitation, making the applicability ofthe findings to other populations debatable. Subgroup analysis bygender was for example not feasible due to the low number of fe-male participants (28 females/236 males) in the whole populationof the meta-analysis and the lack of study specifically investigatingwomen. Two previous studies reported a lower decrease in bloodpressure [53] and no decrease in platelet reactivity [54] in womensupplemented with dietary NO3� compared to men, which suggestthat women may have different metabolic responses to increasedNO3�-NO2�-NO bioavailability.

    Due to the limited number of studies that investigated the effectof dietary NO3� supplementation on exercise tolerance and meta-bolic rate under hypoxic conditions [27,31,42], the meta-analysiscould not confirm that dietary NO3� affects metabolic rate in theseconditions. The rationale for investigating the effect of dietary NO3�

    supplementation in hypoxic condition is that native highlandersexhibit high circulating plasma NO3� and NO2� levels due to an up-regulation of NOS activity that may contribute to their acclimati-zation to environmental hypoxia [55]. Hence, it has been hypoth-esized that subjects not acclimatized to hypoxia may benefit fromenhanced NO bioavailability provided by increased dietary NO3�

    intake, especially when NO synthesis is limited by hypoxia. This issupported by the findings from 2 studies performed in hypoxicconditions that reported VO2 decrease at rest and during moderateintensity exercise following a 6-day beetroot juice supplementation[31], and during heavy intensity exercise after a single ingestion of

    beetroot juice [42]. These results warrant further works, thoughNO3� supplementation failed to facilitate the adaptations to hypoxicenvironment during 6-week of hypoxic training program [27].

    5. Conclusion and perspectives

    Previous meta-analyses already showed that dietary NO3�

    significantly decreases systolic blood pressure [56] and may have atherapeutic potential in patients with cardiovascular diseases andsignificantly improves exercise tolerance in healthy subjects [57].Mechanistically, there were clear evidences from in vitro studiesthat NO increases mitochondrial oxidative efficiency [39,58]. Thepresent meta-analysis supports that increasing NO bioavailabilitythrough dietary NO3� translates into a significantly decreased O2cost during exercise performed in the heavy andmoderate intensitydomains, but not at exercise intensities close to the maximum.These effects of dietary NO3� supplementation may be of interest inpatients with cardiovascular or respiratory diseases that severelylimits exercise capacity, with potential improvement of exercisetolerance that may positively affect their quality of live. On theopposite, there is also a need to determine the factors responsiblefor the lack of effect on VO2 in subjects with high VO2max, as shortterm beetroot juice supplementation is used as an easy, availableand healthy ergogenic aid by athletes. Directions for future studiesshould also include research into the dose and duration of sup-plementation in order to provide recommendation for NO3� sup-plementation specific to each population.

    Acknowledgments

    M.P.C. and J.A. designed the study and conducted the literaturesearch. M.P.C. and J.A. collected, analyzed and interpreted the data.M.P.C., J.A and J.B. wrote the manuscript. J.X.G., G.C. and S.B. revisedsubstantially the manuscript. M.P.C. and J.A had primary re-sponsibility for final content. All authors read and approved thefinal manuscript. The authors do not have any conflicts of interest todisclose.

    Appendix A. Supplementary data

    Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.niox.2016.01.001.

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    Effect of dietary nitrate supplementation on metabolic rate during rest and exercise in human: A systematic review and a me ...1. Introduction2. Methods2.1. Type of studies2.2. Type of interventions2.3. Type of outcome measures2.4. Search strategy2.5. Study selection and data extraction2.6. Data analysis

    3. Results3.1. Study selection3.2. Study characteristics3.3. Risk of bias within studies3.4. Results of individual studies3.5. Synthesis of results3.5.1. Pooled effect size3.5.2. Subgroup analyses3.5.3. Influence of studies with the largest effects3.5.4. Publication bias3.5.5. Heterogeneity and inconsistency

    4. Discussion4.1. Summary of evidence4.2. Limitations

    5. Conclusion and perspectivesAcknowledgmentsAppendix A. Supplementary dataReferences