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Journal of the American College of Nutrition

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Beetroot Juice Does Not Enhance SupramaximalIntermittent Exercise Performance in EliteEndurance Athletes

Mehdi Pawlak-Chaouch, Julien Boissière, Désiré Munyaneza, François-XavierGamelin, Grégory Cuvelier, Serge Berthoin & Julien Aucouturier

To cite this article: Mehdi Pawlak-Chaouch, Julien Boissière, Désiré Munyaneza, François-XavierGamelin, Grégory Cuvelier, Serge Berthoin & Julien Aucouturier (2019): Beetroot Juice Does NotEnhance Supramaximal Intermittent Exercise Performance in Elite Endurance Athletes, Journal ofthe American College of Nutrition, DOI: 10.1080/07315724.2019.1601601

To link to this article: https://doi.org/10.1080/07315724.2019.1601601

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Beetroot Juice Does Not Enhance Supramaximal Intermittent ExercisePerformance in Elite Endurance Athletes

Mehdi Pawlak-Chaoucha, Julien Boissi�erea, D�esir�e Munyanezaa, Francois-Xavier Gamelina, Gr�egory Cuvelierb,Serge Berthoina, and Julien Aucouturiera

aSport, Health and Society, URePSSS–Pluridisciplinary Research Unit, Lille University, Lille, France; bLaboratory of Exercise and Movement,Provincial School of Hainaut (HEPH)-Condorcet, Tournai, Belgium

ABSTRACTObjective: Nitrate (NO3

�)-rich beetroot juice (BR) is recognized as an ergogenic supplement thatimproves exercise tolerance during submaximal to maximal intensity exercise in recreational andcompetitive athletes. A recent study has investigated the effectiveness of BR on exercise perform-ance during supramaximal intensity intermittent exercise (SIE) in Olympic-level track cyclists, butstudies conducted in elite endurance athletes are scarce. The present study aimed to determinewhether BR supplementation enhances the tolerance to SIE in elite endurance athletes.Methods: Eleven elite endurance athletes (age: 21.7±3.7 years, maximal oxygen uptake ð _VO2maxÞ :71.1 ±5.2mL�kg�1�min�1) performed an SIE test until exhaustion following either a 3-day BR sup-plementation (340mg/d) or a placebo (PL) supplementation (<2.5mg/d) in a randomized, singleblind, placebo-controlled, and crossover study. The exercise test consisted of 15-second cyclingexercise bouts at 170% of the maximal aerobic power interspersed with 30-second passive recov-ery periods. Gas exchange was measured during SIE tests as local muscle O2 delivery and extrac-tion were assessed by near infrared spectroscopy.Results: The number of repetitions completed was not significantly different between BR(13.9±4.0 reps) and PL conditions (14.2 ±4.5 reps). BR supplementation did not affect oxygenuptake ( _VO2) during SIE tests (BR: 3378.5 ±681.8mL�min�1, PL: 3466.1±505.3mL�min�1). No sig-nificant change in the areas under curves was found for local muscle total hemoglobin (BR:6816.9±1463.1 arbitrary units (a.u.), PL: 6771.5 ± 3004.5 a.u.) and deoxygenated hemoglobin (BR:6619.7±875.8 a.u., PL: 6332.7 ± 1336.8 a.u.) during time-matched workþ recovery periods from SIEtests following BR supplementation.Conclusions: BR supplementation does not enhance the tolerance to SIE in elite enduranceathletes and affects neither _VO2 nor local muscle O2 delivery and extraction.

Abbreviations: AUC: area under the curve; ATP: adenosine triphosphate; BR: beetroot juice; DBP:diastolic blood pressure; Hb: hemoglobin; HbO2: muscle oxygenated hemoglobin; HHb: muscledeoxygenated hemoglobin; NIRS: near infrared spectroscopy; Mb: myoglobin; NO: nitric oxide;NO3

�: nitrate; NO2�: nitrite; NOx: nitrateþnitrite; PCr: phosphocreatine; PL: placebo; RER: respira-

tory exchange ratio; SBP: systolic blood pressure; SIE: supramaximal intensity intermittent exercise;THb: muscle total hemoglobin; TM: time-matched data, _VE ¼ minute ventilation, _VCO2 ¼ carbondioxide output, _VO2 ¼ oxygen uptake, _VO2max ¼ maximal oxygen uptake; WR: ¼ workrate increment

ARTICLE HISTORYReceived 24 September 2018Accepted 27 March 2019

KEYWORDSDietary nitrate; high aerobicfitness; exercise tolerance;endurance athletes;intermittent exercise

IntroductionNitric oxide (NO) is a ubiquitous signaling molecule thatpossesses the ability to improve vascular function, mitochon-drial efficiency and respiration, glucose homeostasis, andmuscle contractility (1). NO is enzymatically produced bythe nitric oxide synthase that is dependent on L-arginineand oxygen availability and thus becomes limited in hypoxia(2). However, a relevant alternative source of NO hasemerged through the consumption of nitrate (NO3

�)-richvegetables such as lettuce, spinach, and beetroot (3). Dietary

NO3� are absorbed from the small intestine and, after an

entero-salivary recirculation and concentration into the sal-iva, are converted into nitrite (NO2

�) by oral NO3� reduc-

tase bacteria (3). The swallowed salivary NO2� are either

reduced to NO and other nitrogen species in the acidicstomach or absorbed from the intestine, and thus increasethe endogenous circulating NO pool (3).

The consumption of dietary NO3� is recognized as a

nutritional aid to improve tolerance to submaximal to max-imal intensity exercise (4). The increase in exercise tolerance

CONTACT Mehdi Pawlak-Chaouch pawlak.mehdi@orange.fr; medhi.pawlak-chaouch@univ-lille.fr Pluridisciplinary Research Unit, Sport, Health and Society,(URePSSS)–EA7369, Lille University, Eurasport, 413 Avenue Eug�ene Avin�ee, 59120 LOOS, Lille, FranceColor versions of one or more of the figures in the article can be found online at www.tandfonline.com/uacn.

Supplemental data for this article can be accessed at https://doi.org/10.1080/07315724.2019.1601601.

� 2019 American College of Nutrition

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITIONhttps://doi.org/10.1080/07315724.2019.1601601

has been mostly attributed to decreased adenosine triphos-phate (ATP) cost of muscle contraction (5) and improvedoxidative efficiency (6). Some studies reported that NO3

�-rich beetroot juice (BR) supplementation did not changeany indices of mitochondrial coupling and respiratory effi-ciency (7) and did not alter in vivo muscle oxidative effi-ciency in recreationally active males (8). In these studies, theauthors found decreased whole-body and muscle oxygenuptake ( _VO2) during submaximal cycling and isometrichandgrip exercise, respectively (7, 8). Thus, BR supplementa-tion may act through divergent mechanisms than sodiumnitrate (6) by improving skeletal muscle contractileefficiency (5, 7) and/or local tissue perfusion (8, 9).However, a number of studies did not show any change inexercise performance during time-trial tests following NO3

�

supplementation (10–14), particularly in elite enduranceathletes. In situations in which muscle O2 supply may berestricted, Shannon et al. showed that well-trained individu-als can also experience benefits from dietary NO3

� supple-mentation during high-intensity exercise (15).

Moreover, mixed findings have especially been reportedregarding the effect of NO3

� supplementation on supramax-imal intensity intermittent exercise (SIE) performance (9, 10,16–18). This type of exercise is known to recruit a high pro-portion of type II, predominantly glycolytic, muscle fibers(19) that are characterized by lower O2 availability than typeI, highly oxidative, muscle fibers (20). As such, the reductionof NO3

� to NO may be enhanced in type II muscles fibersgiven that it preferentially occurs in both acidic and hypoxicconditions (3). Running time over a single 180-meter sprintwas shown to be improved (16), and we also reportedimproved tolerance to the repetition of supramaximal inten-sity bouts of exercise (� 170% power output at maximaloxygen uptake [ _VO2max]) in moderately fit subjects ( _VO2max

between 40 and 55mL�kg�1�min�1) following dietary NO3�

supplementation (9). However, in contrast to findings dur-ing submaximal intensity exercise, enhanced tolerance toSIE was not related to any decrease in _VO2 or in anaerobicenergy production (9, 17). Apart from improving exercisetolerance, we found an increase in local muscle total hemo-globin during SIE after 3-day BR supplementation (9). Bybetter maintaining O2 delivery to the working muscle, NO3

�

supplementation likely delays the depletion of musclephosphocreatine (PCr) and the accumulation of adenosinediphosphate and phosphate ion metabolites (21) and pro-motes PCr resynthesis, dependent on oxidative phosphoryl-ation (22). In combination, these effects can attenuate thedevelopment of muscle fatigue and contribute to sustainpower output between supramaximal intensity exercisebouts. To date, no investigation has assessed local muscle O2

delivery and extraction in elite endurance athletes conduct-ing SIE tests after BR supplementation.

There is mounting evidence suggesting that the level ofaerobic fitness of athletes modulates the effects of NO3

�

supplementation on performance (23, 24). Porcelli et al.demonstrated that the magnitude of increase in exercise per-formance during a 3000-meter running race time trial wasinversely correlated to _VO2max; with no benefit for athletes

with _VO2max above 60mL�kg�1�min�1. Conversely, moststudies that reported enhanced performance were conductedin non-endurance athletes or moderately trained subjects( _VO2max � 50mL�kg�1�min�1) (5, 9, 25–30). The reasonsfor a lower effectiveness are still unclear but may be relatedto characteristics of athletes with high _VO2max; such asalready high synthesis of NO through the enzymatic path-way (31) and a high proportion of type I muscle fibers,which are less sensitive to an increase in NO bioavailabilitythan type II muscle fibers (20).

Nevertheless, dietary NO3� supplementation may be of

great interest to endurance athletes who will engage in high-intensity training and/or events. Although mean exerciseintensity is submaximal in endurance sports, the time spentbeyond power output corresponding to _VO2max can exceed15% of the total race duration in activities such as cycling ortriathlon (32). Moreover, the time spent at these supramaxi-mal intensities is distributed into short bouts of exerciseacross the duration of the event (33). The exercise perform-ance may be impacted by the capacity to maintain efforts insuccessive high-intensity exercise intervals over a long timeperiod (34). Furthermore, middle-distance athletes (800 to5000–10,000 meters), who are also characterized by high_VO2max; select pacing strategies during running competitionevents so that they regularly run at speeds well above the_VO2max intensity (35). Interval training protocols at supra-maximal intensity allow to maximally strain aerobic andanaerobic metabolism (36) and to enhance physiologicaldeterminants of exercise performance even in athletes whohave already elicited cardiorespiratory adaptations to chronicexercise training (35, 37, 38).

Our purpose was therefore to determine whether dietaryNO3

� supplementation enhances tolerance to SIE in eliteendurance athletes having high aerobic fitness ( _VO2max >65mL�kg�1�min�1). We hypothesized that dietary NO3

�

supplementation would increase plasma nitrateþ nitrite(NOx) levels without improving tolerance to exercise.

Material and methods

Population

Subjects’ characteristics are presented in Table 1. Subjectswere contacted in track and field and triathlon clubs andwere all engaged in intense endurance exercise trainingand competitive running or triathlon. Eleven subjects wereinitially recruited for the study and two subjectswere excluded after completing the preliminary visit becausethey did not meet the criteria for inclusion ( _VO2max >65mL�kg�1�min�1). Subjects were not taking any nutritional

Table 1. Characteristics of the subjects included in the study (n¼ 9).

Parameters Mean ± SD

Age (years) 21.7 ± 3.7Body mass (kg) 66.8 ± 8.5Height (m) 1.80 ± 0.07Body fat (%) 11.1 ± 2.4Maximal aerobic power (W) 327 ± 30_VO2max (mL�kg�1�min�1) 71.1 ± 5.2_VO2max ¼ maximal oxygen uptake.

2 M. PAWLAK-CHAOUCH ET AL.

supplement or medication known to affect NO metabolism.All subjects were informed about the study protocol and itspotential risks and benefits and gave written consent to par-ticipate in this study. Subjects were concealed to the trueaim of study and were told that the purpose of trials was tocompare the ergogenic effects of two beverages. The proto-col of the study complied with the Declaration of Helsinkiand was approved by the Institutional Ethics Committee ofthe Haute Ecole Provinciale du Hainaut.

Incremental maximal exercise test

At least one week before the first experimental visit, subjectsperformed an incremental exercise test until volitionalexhaustion to ensure their eligibility for the study. _VO2max

and power output at _VO2max were measured during a gradedexercise test until volitional exhaustion performed on a mech-anically braked cycle ergometer (894E, Monark Exercise TM).The test was initiated at 100W for 3minutes and increasedby 35-W increments every 2minutes. Subjects were invited tomaintain a pedaling frequency of 80 rpm and were stronglyencouraged throughout the test to perform a maximal effort.We considered that _VO2max was achieved when at least threeof the following criteria were established: volitional exhaus-tion, theoretical maximal heart rate (220 � age ± 10beats�min�1), respiratory exchange ratio (RER; _VCO2/ _VO2)above 1.1, and a _VO2 plateau (� 2.1mL�kg�1�min�1)between the last two stages. Maximal aerobic power output(Waerobic max) was determined as the sum of the power outputduring the last stage fully completed (Wfinal) and the workrate increment (WR) multiplied by the fraction of time spent(t) in the final unfinished stage as follows:

Waerobic max ¼ Wfinal þ t120

�WR

� �

Dietary supplementation conditions

Subjects were supplemented either with dietary NO3� with

BR with an average nitrate content of 680mg�L�1 orapple–black currant juice used as a placebo (PL) witha nitrate content < 5mg�L�1 in a randomized, single blind,placebo controlled, crossover study. We used a randomnumber table to assign the order of supplementation condi-tions to each participant. The experimenter engaged in therandomization process did not take part in the experimentalvisits. Because the placebo juice differs in taste, smell, andappearance compared to BR supplementation, the purposeof the study was communicated to the participants as thecomparison of exercise performance and physiologicalresponses after the consumption of two vegetable beverages.There was a minor difference in the energy provided by BR(185 kcal) and placebo (225 kcal). Even although beetroot isrich in antioxidants, polyphenols, and flavonoids, nitrateappears to be the active compound responsible for thephysiologic effects following BR supplementation. Lansleyet al. found no reduction in _VO2 during moderate- andsevere-intensity exercise in healthy, recreationally active sub-jects following nitrate-depleted BR using an ion-exchange

resin that selectively removes NO3� ions (26). The two bev-

erages were purchased from Pajottenlander TM. Drinkswere distributed by an independent technician, not involvedin the process of exercise testing. In the morning of the twodays preceding each experimental visit, subjects ingestedeither 500mL of BR or 500mL of apple–black currant juice.On the day of each experimental visit, subjects wereinstructed to ingest 500mL of BR or placebo 2 hours beforethe time of their appointment at the laboratory so that thestart of SIE tests corresponded to plasma nitrite peak.Pharmacokinetic data suggest that plasma nitrite will peak2.5 to 3 hours after ingestion of a single dose of BR (39).Subjects were instructed to refrain from caffeine and alcohol6 and 24 hours before the experimental visits, respectively.Participants were not asked to refrain from consumingNO3

�-rich food products, but they were instructed to repli-cate their food intake over the supplementation daysbetween the two conditions and to complete a daily fooddiary for each supplementation period. In addition, subjectswere finally instructed not to brush their teeth or to usemouthwash for 3 hours after each BR or placebo intake (40).

SIE tests

After the preliminary visit, subjects performed cycling SIEtests during the two sessions 180minutes after the last doseof either ingested dietary nitrate or placebo supplementation.The exercise tests were completed on the same cycle ergom-eter than during the preliminary visit and separated by atleast 2weeks. Subjects warmed up for 5minutes at 50% ofmaximal aerobic power output measured during the prelimin-ary visit and were instructed to maintain a cycling cadenceof 70 rpm. After a 2-minute recovery period, subjects startedthe test consisting of work period repetitions composed asfollows: 15-second cycling bout at 170% of maximal aerobicpower output interspersed with 30-second passive recoveryperiods. Before the test, subjects were instructed to reach asquickly as possible the targeted pedaling frequency of 90 rpmduring each 15-second cycling exercise bout. Subjects startedpedaling after a 3-second countdown by one of the experi-menters, and when they reached 50 rpm the resistance corre-sponding to 170% of maximal aerobic power output wasautomatically applied with the Monark Software (MonarkAnaerobic Test, Monark Exercise TM). Subjects recoveredpassively between each work period. The test was stoppedwhen subjects indicated volitional exhaustion or when theywere no longer able to maintain a pedaling frequency above87 rpm during the 5 last seconds of a 15-second cycling bout.

Gas exchange measurement

_VO2; carbon dioxide output ( _VCO2), RER (RER¼ _VCO2/_VO2), and minute ventilation ( _VE) were measured breathby breath through a gas exchange measurement system(K4b2, COSMED TM) during the graded maximal exercisetest and SIE tests. Before each test, the gas analyzer wascalibrated with ambient air and a gas mixture of knownconcentration (O2: 16%, CO2: 5%), and the turbine

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 3

flowmeter was calibrated with a 3 L syringe. Gas exchangedata were recorded on a computer for later analysis. Heartrate was continuously recorded with a heart rate monitor(S810, Polar TM).

Local muscle parameters

The changes in local muscle microvascular oxygenation,deoxygenation, and total-hemoglobin volume of the vastuslateralis muscle were continuously measured during SIE testsby near infrared spectroscopy (NIRS; OxyMon, ArtinisMedical Systems TM). Briefly, the NIRS system measuresthe change in light absorption by hemoglobin (Hb) andmyoglobin (Mb), which is related to Hb and Mb O2 satur-ation. The two wavelengths emitting (780 and 850 nm) andreceiving optodes were spaced out of 4 cm and were placedat midway between the lateral condyle of the knee and thegreater trochanter of the femur. Optodes were taped to theskin with adhesive strapping, covered with a dense blackcloth to minimize exogenous light contamination, andwrapped with an elastic bandage to avoid any movementduring the exercise tests. The location of the optodes wasmarked with a permanent pen during the first experimentalvisit to place them at the same location during the secondexperimental visit. NIRS signal acquisition was performed at10Hz with the Oxysoft software (Oxysoft, Artinis MedicalSystems TM), and data were exported at 1Hz for later ana-lysis. Three indexes of muscle oxy-, deoxy-, and total-hemo-globin volume were derived from NIRS measurements ofthe vastus lateralis muscle: changes from the resting baselinein muscle oxygenated hemoglobin (HbO2) and muscledeoxygenated hemoglobin (HHb) were respectively estimatesof muscle oxygenation and fractional O2 extraction; muscletotal hemoglobin (THb) was calculated as the sum of HbO2

and HHb and was an estimate of the change in musclemicrovascular blood volume.

Blood pressure measurement

Upon their arrival at the laboratory, subjects rested for10minutes in supine position on a medical exam table.Resting systolic blood pressure (SBP), diastolic blood pres-sure (DBP), and heart rate were measured in duplicate withan electronic sphygmomanometer (IntelliSense, Omron TM)and then were averaged for analysis.

Blood sampling and assessment

After blood pressure measurements, blood sampling wasperformed through a catheter inserted in an antecubitalvein. A first sample (5mL) was collected 15minutes beforeSIE tests in an ethylenediaminetetraacetic acid (EDTA)-content tube with subjects in a semi-recumbent position.Twenty-five minutes after the end of exercise tests, a secondsample was drawn with the same procedure. Samples werecentrifuged at 3600 g and 4 �C for 10min, and plasma wasaliquoted and stored at �80 �C for later analysis.

Plasma NOx concentrations were determined by theGriess method with a commercial kit (Cayman NO3

� colori-metric assay kit, Bertin Pharma TM).

Data analysis

Given that each subject completed a number of repetitionsthat were different between BR and PL conditions, theexperimental condition with the lowest number of repeti-tions completed was used as the trial reference and wascompared to time-matched data of the other experimentalcondition with the highest number of repetitions. The time-matched gas exchange and NIRS variables comparedbetween the two conditions are denoted TM throughout theResults and Discussion sections. Mean gas exchange valueswere calculated over the whole exercise duration, only TMwork bouts, only TM recovery bouts, and combined TMwork and recovery periods, respectively. The amplitude ofchange for _VO2; _VCO2; and _VE (D _VO2; D _VCO2; andD _VE; respectively) between work and recovery periods dur-ing TM repetitions was also calculated.

All NIRS variables were expressed as relative changesfrom baseline values. Baseline values for HbO2, HHb, andTHb were measured during the last 30 seconds of a2-minute resting period with subjects in a seated positionbefore the warm-up for the exercise test. The maximal val-ues for HHb and THb, and the minimal and maximal valuesfor HbO2 (HbO2min and HbO2max, respectively) were deter-mined during each experimental condition. The areas underthe curves (AUC) for HbO2 (AUC-HbO2þ), HHb (AUC-HHb), and THb (AUC-THb) were calculated by the trapez-oidal method over the duration of combined TM work andrecovery periods. The AUC were calculated to provide anintegrated index of muscle oxy-, deoxy-, and total-hemoglo-bin volume. During exercise, changes in HbO2 occur belowthe baseline level; the AUC for HbO2 was also determinedbelow the baseline level (AUC-HbO2-).

Statistical analysis

Statistical analyses were run with R software (R version 3.2.5,R Foundation for Statistical Computing). Data are expressedas mean± standard deviation (SD) unless otherwise stated.Normal distribution of the data was assessed by theKolmogorov–Smirnov test. A bilateral Student paired t testwas used to compare the dependent variables (gas exchangevalues, NIRS-derived indexes, and exercise performanceparameters) from SIE tests between the experimentalconditions (independent variables: BR supplementation vs.placebo). Plasma NOx levels were analyzed by two-wayanalysis of variance for repeated measures to comparethe experimental conditions (BR supplementation vs. placebo)before and after the exercise test. Post-hoc analysis was thenperformed for dependent samples, as appropriate. Pearson’sproduct-moment correlation coefficients were calculatedto assess the relationships between the change in NOx

and the changes in exercise tolerance and physiologicalparameters. Statistical significance was set at p< 0.05.

4 M. PAWLAK-CHAOUCH ET AL.

Results

Plasma NOx levels

Plasma NOx levels were significantly higher in the BRcondition compared to the PL condition before (BR:91.05 ± 30.01lM vs. PL: 21.41 ± 7.59 lM; p< 0.01) and afterSIE (BR: 78.23 ± 14.67lM vs. PL: 20.17 ± 6.17 lM; p< 0.01).

Exercise performance

Individual number of repetitions is displayed in Figure 1.The number of work periods completed was not signifi-cantly different between the two conditions (BR: 13.9 ± 4.0reps, PL: 14.2 ± 4.5 reps). The targeted power outputrepresenting 170% of maximal aerobic power output was555.3 ± 50.3 W. Mean power output was 579.2 ± 57.7 Wduring the BR condition and 578.9 ± 54.3 W during the PLcondition. There was no difference for total work performedduring the two conditions (BR: 121 ± 38 kJ, PL: 121 ± 29 kJ).The number of work bouts completed was not significantlyassociated with plasma NOx levels in any of the two condi-tions both before (BR: r ¼ �0.05, PL: r ¼ �0.44) and after(BR: r ¼ �0.26, PL: r ¼ �0.45) the SIE test. The order ofthe experimental sessions did not significantly affect exerciseperformance (first session: 13.2 ± 3.7 reps, second session:14.8 ± 4.6 reps).

Gas exchange and heart rate

There were no significant differences for _VO2; _VCO2; and_VE between the BR and PL conditions for the wholeexercise, the first three work and recovery periods, and TMwork and recovery periods (see Figure 2). Heart rate was

also similar between the two conditions for the wholeexercise (BR: 159.9 ± 9.0 bpm, PL: 156.9 ± 15.4 bpm) and forTM periods (BR: 170.4 ± 14.7 bpm, PL: 172.0 ± 7.1 bpm).There was no significant association between the changesin plasma NOx levels and the changes of _VO2 in any ofthe two conditions both before (BR: r¼ 0.12, PL: r¼ 0.19for TM workþ recovery periods) and after (BR: r¼ 0.244,PL: r¼ 0.325 for TM workþ recovery periods) SIE test. Allcorrelations between plasma NOx levels and _VO2 changesare reported in Supplemental online Table 3. Results for gasexchange parameters and heart rate during exercise testsperiods between BR and PL conditions are shown inSupplemental online Table 1.

NIRS-derived parameters and resting blood pressure

Results from NIRS measurements are displayed in Table 2.At baseline, HbO2, HHb, and THb were not significantlydifferent between the two conditions. During exercise, themaximal values of HbO2, HHb, and THb were not signifi-cantly different between the two conditions. AUC-HbO2þand AUC-HbO2-, AUC-HHb, and AUC-THb did not differbetween the BR condition and PL. The changes in plasmaNOx levels were not significantly associated with the changesin fractional O2 extraction (before SIE test, BR: r¼ 0.37, PL:r¼ 0.39; after SIE test, BR: r¼ 0.34, PL: r ¼ �0.03) andmuscle microvascular blood volume (before SIE test, BR:r¼ 0.22, PL: r¼ 0.32; after SIE test, BR: r¼ 0.15, PL: r ¼�0.19) during TM workþ recovery periods in any of thetwo conditions. All correlations between plasma NOx levelsand changes in NIRS-derived parameters are reported inSupplemental online Table 3. There was no significant effect

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Figure 1. Individual data points for the number of repetitions completed before volitional exhaustion during supramaximal intensity intermittent exercise testsfollowing a placebo and a beetroot juice supplementation. Red squares indicate the mean number of work periods completed for each condition.

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 5

of BR supplementation on SBP, DBP, and heart rate at rest(Supplemental online Table 2).

Discussion

The main finding of our study is the lack of improvementin tolerance to SIE after dietary nitrate supplementation inathletes with a high level of aerobic fitness ( _VO2max >

65mL�kg�1�min�1). The present study, to our knowledge, isthe first to investigate the exercise tolerance to SIE and tomeasure gas exchange and local muscle O2 delivery andextraction in elite endurance athletes. Regardless of the aer-obic fitness status of participants, a few studies have ques-tioned whether NO3

� supplementation increases toleranceto SIE. These studies showed either an impaired (17),unchanged (10, 16–18), or improved performance (9, 18).

High aerobic fitness is likely a major factor contributingto the lack of effect of NO3

� supplementation on exerciseperformance (23, 24). Previous studies showed that 1 day(11–14, 16) and 6 days of dietary NO3

� supplementation(10, 13, 23) did not improve exercise performanceduring running (11, 12, 16, 23) and cycling time trials (10,13, 14) in endurance athletes with _VO2max above�60mL�kg�1�min�1. Few nutritional supplementations haveshowed good benefits for exercise performance in elite

athletes that are already well adapted to sport events dueto chronic training adaptations (41). Investigations thataddress the effectiveness of dietary NO3

� supplementationduring supramaximal intensity exercise in elite enduranceathletes are rare to date, with no improved exercise perform-ance during 180-meter running time trials in elite cross-country skiers (16) and during repeated sprint capacity

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Figure 2. Bar plots of gas exchange and heart rate parameters during supramaximal intensity intermittent exercise tests following a placebo (white bars) and abeetroot juice supplementation (grey bars). Data were calculated over the whole duration (Whole session), the first three work and recovery periods (First 3 bouts),and TM-work and recovery periods (Last 3 bouts) for _VO2 (A), _VCO2 (B), _VE (C), and heart rate (D).

Table 2. Effects of a 3-day dietary nitrate supplementation (beetroot juice) onnear infrared spectroscopy–derived parameters of the vastus lateralis muscleduring supramaximal intensity intermittent exercise tests.

Beetroot Juice Placebo

HbO2 Baseline 39.3 ± 14.1 40.3 ± 14.1Maximal 14.3 ± 7.9 12.7 ± 3.8TM-AUCþ 1710.8 ± 1083.7 1911.1 ± 1026.3Minimal �14.5 ± 6.4 �15.0 ± 7.3TM-AUC� 3178.5 ± 1597.5 4013.3 ± 2166.7

HHb Baseline 45.1 ± 18.2 44.9 ± 17.7Maximal 25.6 ± 12.7 21.8 ± 6.5TM-AUC 6619.7 ± 875.8 6332.7 ± 1336.8

THb Baseline 83.0 ± 33.4 85.2 ± 31.2Maximal 15.4 ± 12.3 15.6 ± 5.2TM-AUC 6816.9 ± 1463.1 6771.5 ± 3004.5

Values are mean ± SD.AUC¼ area under the curve; HbO2 ¼ oxygenated hemoglobin;HHb¼ deoxygenated hemoglobin; THb¼ total hemoglobin, TM¼ denotes time-matched comparison for 3 successive workþ recovery periods; TM-AUCþ andTM-AUC�¼ areas under curves calculated for HbO2 data above and below theresting baseline values, respectively; TM-AUC ¼ areas under curves calculated forHHb and THb data above the resting baseline values, respectively.

6 M. PAWLAK-CHAOUCH ET AL.

(6� 20-second sprints, recovery 100 seconds) in elite cyclists(10). It is striking that, with the same exercise protocolas in the present study, team sports athletes with moderateaerobic fitness ( _VO2max < 55mL�kg�1�min�1) had a 20%increase in the mean number of repetitions completed whensupplemented with dietary NO3

� compared to PL (9). Inthis exercise protocol, we fixed the recovery time at30 seconds, which is sufficiently long to allow the PCr poolto be resynthesized and the lactic acid accumulation to belimited (35). When subjects performed repeated sprints with30 seconds of recovery between each sprint, the power out-put was maintained by ATP that was derived mainly fromPCr degradation and an increased contribution of aerobicmetabolism (42). PCr recovery kinetics have been shown tobe dependent on O2 availability in human skeletal muscle(43). The reasons for the absence of change in exercise toler-ance in the present study may be related to characteristics ofelite endurance athletes such a high proportion of type Imuscle fibers (44), greater skeletal muscle capillary density(45), and increased oxidative capacity (46). Tesch et al.showed a similar PCr degradation between type I and typeII muscle fibers in vastus lateralis muscle in active men fol-lowing 30 maximal voluntary knee extensions (47).However, after 60 seconds of recovery, type I muscle fibershad a significant greater level of PCr relative to the initiallevel than type II muscle fibers (47). These results suggestthat elite endurance athletes may already have an optimalrate of PCr resynthesis during recovery periods that couldnot be affected by dietary NO3

� supplementation.Hern�andez et al. well showed that NO3

� supplementationincreased force production in type II, predominantly glyco-lytic, muscle fibers, but not in type I, highly oxidative, musclefibers in mice (48). The same authors reported an increase inthe expression of the sarcoplasmic reticulum Ca2þ-handlingproteins only in type II muscle fibers of NO3

� supplementedmice. Further study is required to address whether NO3

�

supplementation elicits comparable specific physiologicalresponses of type II muscle fibers to exercise in humans. Onepurpose of the present study was therefore to investigate sub-jects with high aerobic fitness who are known to have a highproportion of type I muscle fibers and a small proportion oftype II muscle fibers (44). The unaltered exercise tolerance inthe present study provides support to the hypothesis thatincreased NO bioavailability may have selective effectsin subjects with a high proportion of type II muscle fibers(9, 49) or during exercise that elicits a high recruitment oftype II muscle fibers (20, 50).

There may be a need for a higher NO3� dose than what

we used to elicit significant physiological effects and subse-quent increase in exercise tolerance in athletes with highaerobic fitness (12, 23). Such athletes have indeed beenshown to have already high plasma NOx levels (31, 51), andNO3

� doses that are effective in populations with lower aer-obic fitness may be ineffective to increase NOx levels in thispopulation. Indeed, highly trained rowers improved 2000-meter rowing performance when supplemented with8.4mmol, as did not with 4.2mmol of NO3

� (52). Nitratesupplementation in the present study (5.2mmol/d�1) being

only slightly higher than the lowest dose used by Hoonet al. may have been insufficient to elicit significant physio-logical effects (52). It is important, however, to note thatwhen using NO3

� dose as high as 19.5mmol either acutelyor during 8 days, Boorsma et al. showed no effect on 1500-meter time trial performance of elite middle-distance run-ners (12). Furthermore, the duration of NO3

� supplementa-tion being ingested could be considered. Vanhatalo et al.showed that maximal aerobic power output of healthy, butnot highly trained subjects, was significantly improved rela-tive to PL after 15 days of NO3

� supplementation, whereasthere were no significant differences with a 5-day or acutesupplementation (53). Studies with supplementation dur-ation of at least 15 days have to our knowledge not beenconducted in athletes with high aerobic fitness. However,studies comparing 6 to 8 days’ NO3

� supplementation inathletes with high _VO2max showed no additional benefit (12,13), and NO3

� and NO2� plasma levels were not greater

after the longer NO3� supplementation duration than the

shorter one (13). In addition, a recent meta-analysis showedthat features of the NO3

� supplementation (the dose ofNO3

� supplementation, the number of days of supplementa-tion, and the total amount ingested of NO3

�) were not asso-ciated with changes in physical performance in both non-athlete and athlete individuals (54). In all, further research isrequired to establish the real sufficient dose of NO3

� beingingested, if any, in order to enhance the responsiveness toNO3

� supplementation in elite endurance athletes.Regarding the present study, plasma NOx levels after diet-

ary NO3� supplementation were lower than in previous stud-

ies in subjects with high aerobic fitness (11, 12, 16, 55).However, Porcelli et al., who also ultrafiltrated plasma sam-ples and used the Griess assay, found levels similar to thosethat we report in the PL and NO3

� supplemented conditions(23). Of note, many studies do not report ultrafiltration ofplasma samples, and proteins may interfere with NO3

�/NO2

�, resulting in erroneously high NOx values (56). Finally,a number of studies have reported that resting plasma NO2

�

levels were similar in elite athletes and non-athletes (23, 55)and that acute dietary NO3

� supplementation in elite athleteswith a dose similar to that used in non-athletes (614mg) wasable to elicit significant increases in plasma NO3

�/NO2�, but

no change in performance (11, 16).A second and important finding of the present study was

that _VO2 and local muscle oxy-, deoxy-, and total-hemoglo-bin volume were unaffected by NO3

� supplementation.These findings are in contrast to the repeatedly reporteddecrease in _VO2 during submaximal intensity exercise insubjects with lower _VO2max (5, 25, 26, 29, 57, 58).Unchanged _VO2 was previously reported in studies investi-gating the effects of NO3

� supplementation during SIE (9,17, 18). The percentage of _VO2max elicited in the presentstudy (�75%) was similar to that reported by Martin et al.,who showed no improvement in exercise tolerance withNO3

� supplementation (17). In contrast, the team sportsplayers that we previously investigated reached on average85% _VO2max and had a significant increase in exercise toler-ance with NO3

� supplementation (9). In the latter study, we

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 7

also observed that NO3� supplementation was associated

with significantly higher _VE and lower _VO2 and _VCO2 fluc-tuations. These responses to NO3

� supplementation werenot present in the current study. Even though it is unclearhow these physiological changes contributed to increasedexercise tolerance in previous study, their absence in thepresent study provide additional evidence that increasingNO bioavailability has different effects in endurance athleteswith high aerobic fitness levels. We, however, cannot fullyexclude that NO3

� supplementation would be effective inelite endurance athletes during intermittent exercise withwork:recovery ratio periods that would elicit a higher per-centage of _VO2max (18).

It has been established that high-intensity intermittentexercise heavily recruits type II muscle fibers (19), which aremore sensitive to fatigue than type I muscle fibers (59). BRsupplementation improves blood flow distribution towardtype II muscle fibers (60) and increases microvascular O2

pressure in type II muscle fibers during exercise in rats (61).It is thus hypothesized that dietary NO3

� supplementationmay improve exercise tolerance during high-intensity inter-mittent exercise by better matching blood flow to metabolicrate across the working muscle (62). In accordance with thisperspective, we have previously reported that 3-day BR sup-plementation, similar to that used in the present study,enhances tolerance to exercise at supramaximal intensitywith increased microvascular total-hemoglobin in the work-ing muscle (9). However, to date, there has been no investi-gation of local muscle O2 delivery and extraction in athleteswith high _VO2max and a low proportion of type 2 musclefibers supplemented in dietary NO3

� during SIE. UsingNIRS-derived indexes, we show that O2 extraction and localblood volume in muscle vastus lateralis were unaltered dur-ing SIE. Furthermore, the lack of differences for HHbparameters, in conjunction with similar changes in HbO2

and THb between BR and PL conditions, provides add-itional supports that dietary NO3

� did not affect musclemicrovascular perfusion in elite endurance athletes duringSIE. Our present findings were supported by a recent studythat reported no effect of single dose of BR supplementationon muscle fractional O2 extraction and muscle _VO2 duringsustained isometric exercise in moderately trained subjects(8). In the latter study, the lack of effect of BR supplementa-tion was evident even when blood flow was obstructed toworking muscles (8).

Together, the absence of change in exercise tolerance andphysiological responses after BR supplementation in eliteendurance athletes could be a consequence of many adapta-tions resulting of chronic training such as enhanced NObioavailability by an upregulation of NO synthase activity(31, 63), greater skeletal muscle capillary density (45),increased content of skeletal muscle Ca2þ-handling proteins(64), and a lower proportion of type II muscle fibers (44).

Study considerations

The low number of subjects can be considered as a limita-tion of our study. We cannot exclude that a study including

a higher number of subjects would have yielded differentresults. However, rather than including a large numberof subjects, we recruited a well-characterized populationof healthy young men based on _VO2max above65mL�kg�1�min�1 and participation to endurance competi-tive sports.

Another limitation of the study was the small dose ofnitrate (5.2mmol/d�1) used in the present study. It has beensuggested that the minimal effective dose required toenhance exercise performance in most individuals is�5mmol of NO3

� per serving (65, 66). However, a recentmeta-analysis reported that the dose of NO3

� supplementedand the total amount of NO3

� ingested were not associatedwith changes in exercise performance in non-athletes andathletes (54). Thus, further research is needed to determinewhether higher NO3

� intake than in our study is requiredin athletes with high _VO2max to improve the tolerance toSIE, although few BR products intended for athletes effect-ively provide � 5mmol of NO3

� per serving (66)

Conclusions

Three days of NO3�-rich BR supplementation (5.2mmol/

d�1) did not increase the tolerance to SIE in elite enduranceathletes with high _VO2max; and did not affect _VO2 and localmuscle O2 delivery and extraction. This finding providesadditional support to the hypothesis that BR supplementa-tion is less effective to improve performance in endurance-trained athletes, even in the case of exercise modalities thatare known to promote the effectiveness of dietary NO3

�

supplementation by recruiting a greater proportion of NO-sensitive type II muscle fibers.

Acknowledgements

We are grateful to the athletes who took part to the study. We thankthe nurses at the Haute Ecole Provinciale du Hainaut (Tournai,Belgium) for their assistance during the experimental protocol.

Authors J.A., J.B., S.B., and M.P.-C. conceived and designed theresearch; authors J.A., J.B., F.-X.G., D.M., G.C., and M.P.-C. conductedthe research; authors J.A., J.B., D.M., and M.P.-C. analyzed the data orperformed statistical analysis; authors J.A., J.B., F.-X.G., G.C., D.M.,S.B., and M.P.-C. wrote the article. M.P.-C. and J.A. had primaryresponsibility for final content. All authors read and approved thefinal manuscript.

Disclosure Statement

The authors do not have any conflicts of interest to disclose. No fund-ing was received for this study.

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