Effects of Dietary Nitrate Supplementation on Performance during Single and Repeated Bouts of Short-Duration High-Intensity Exercise: A Systematic Review and Meta-Analysis of Randomised Controlled Trials

Inorganic nitrate (NO3−) has emerged as a potential ergogenic aid over the last couple of decades. While recent systematic reviews and meta-analyses have suggested some small positive effects of NO3− supplementation on performance across a range of exercise tasks, the effect of NO3− supplementation on performance during single and repeated bouts of short-duration, high-intensity exercise is unclear. This review was conducted following PRISMA guidelines. MEDLINE and SPORTDiscus were searched from inception to January 2023. A paired analysis model for cross-over trials was incorporated to perform a random effects meta-analysis for each performance outcome and to generate standardized mean differences (SMD) between the NO3− and placebo supplementation conditions. The systematic review and meta-analysis included 27 and 23 studies, respectively. Time to reach peak power (SMD: 0.75, p = 0.02), mean power output (SMD: 0.20, p = 0.02), and total distance covered in the Yo-Yo intermittent recovery level 1 test (SMD: 0.17, p < 0.0001) were all improved after NO3− supplementation. Dietary NO3− supplementation had small positive effects on some performance outcomes during single and repeated bouts of high-intensity exercise. Therefore, athletes competing in sports requiring single or repeated bouts of high-intensity exercise may benefit from NO3− supplementation.


Introduction
Inorganic nitrate (NO 3 − ) has been conventionally considered an environmental carcinogen and inert end-product of endogenous nitric oxide (NO) oxidation [1]. More recent research challenges these assertions and has revealed various potential health benefits afforded by increased dietary NO 3 − intake [2]. Over the last couple of decades, dietary NO 3 − supplementation has emerged as a potential nutritional strategy to improve exercise performance in healthy and moderately trained individuals [3,4]. The ergogenic effects of NO 3 − supplementation have been attributed to its stepwise reduction to nitrite (NO 2 − ) and the subsequent reduction of NO 2 − to NO [2,5]. Although initially recognised for its vasodilatory properties [6], it is now appreciated that NO can positively modulate a plethora of physiological responses in skeletal muscle [7][8][9], the conflation of which is likely to underpin improved exercise performance following dietary NO 3 − supplementation [5]. Initial studies assessing the potential efficacy of NO 3 − supplementation to enhance physiological and performance responses during exercise revealed improvements in exer-cise economy and exercise tolerance [10][11][12]. These improvements in endurance exercise performance parameters after NO 3 − supplementation were initially linked to a lower adenosine triphosphate (ATP) cost of muscle force production (improved contractile efficiency), an associated blunting in the perturbation to high-energy phosphate substrates and metabolites [13], and to a lower mitochondrial adenosine diphosphate/oxygen ratio (P/O ratio; a lower O 2 cost of ATP resynthesis), reflecting improved mitochondrial respiratory efficiency [14]. However, the mechanisms by which NO 3 − supplementation can improve exercise economy and endurance exercise performance are still to be resolved in human skeletal muscle [15,16].
Following on from the initial human studies, experiments conducted using murine models indicated potential fibre-type-specific effects of NO 3 − supplementation on physiological responses [17]. Indeed, NO 3 − supplementation was initially reported to increase calcium (Ca 2+ ) handling proteins and evoked force production in type II skeletal muscle, but not slow-twitch (type I) skeletal muscle, in mice [18]. Subsequently, NO 3 − supplementation increased hindlimb blood flow in exercising rats, with this additional blood flow shunted towards more fast-twitch (type II) muscle fibres [19]. The potential for enhanced efficacy of NO 3 − supplementation to improve physiological and performance responses in murine type II muscle is consistent with data from human studies demonstrating enhanced pulmonary O 2 uptake ( . VO 2 ) and muscle deoxyhaemoglobin + deoxymyoglobin kinetics in exercise settings that evoke greater type II muscle fibre recruitment compared to exercise settings that evoke mostly type I muscle fibre recruitment [20]. Moreover, cross-sectional data have revealed that NO 3 − supplementation is less likely to improve exercise economy and endurance performance as aerobic fitness increases [21], an effect that has been attributed, at least in part, to a lower % and proportional recruitment of type II muscle fibres in endurance-trained participants with a more aerobic phenotype [22]. On this basis, NO 3 − supplementation may have greater potential as an ergogenic aid in exercise settings which evoke greater type II muscle fibre recruitment.
It is well documented that type II skeletal muscle fibres are recruited in an intensitydependent manner, with greater recruitment of type II muscle fibres at higher exercise intensities [23][24][25]. In addition, the reduction of NO 2 − to NO is enhanced in conditions of acidosis and hypoxia [26][27][28]. The partial pressures of O 2 (PO 2 ) and pH are lower in contracting type II than type I muscles [29,30] and progressively decline with increasing exercise intensity [31]. Therefore, high-intensity exercise, which is supramaximal with regards to the power output required to elicit . VO 2max , and evokes significant recruitment of type II muscle fibres and declines in muscle pH and PO 2 , appears to have greater potential to elicit an ergogenic effect from NO 3 − supplementation compared to continuous submaximal endurance exercise. There is also evidence to suggest that NO 3 − supplementation is more effective at improving physiological and functional responses at higher, compared to lower, movement velocities [32,33]. In addition, NO 3 − supplementation has been reported to increase the peak contractile velocity of, and power output generated by, contracting skeletal muscle [33,34], and to lower the time taken to achieve peak power output [35,36]. Collectively, these improvements in skeletal muscle contractile function after NO 3 − supplementation would be expected to translate into enhanced single and repeated sprint performances. However, whilst there is some evidence to support an ergogenic effect of NO 3 − supplementation on single and repeated bouts of short-duration large muscle mass exercise in humans (e.g., [37,38]), the existing evidence basis is equivocal (e.g., [39][40][41]). In part, these interstudy discrepancies may be attributable to disparate NO 3 − supplementation and high-intensity exercise protocols, which complicates interpretation of the ergogenic potential of NO 3 − supplementation for high-intensity exercise. Although the effects of NO 3 − supplementation on performance in a variety of exercise performance tests have been systematically reviewed and have undergone meta-analyses before [42][43][44][45][46][47][48], these have not yet considered the effects of NO 3 − supplementation on single and repeated bouts of short-duration large muscle mass exercise in humans. This is important to address to help improve understanding of the exercise settings in which NO 3 − Antioxidants 2023, 12, 1194 3 of 20 supplementation is ergogenic and to inform recommendations for NO 3 − supplementation to improve exercise performance. Therefore, the purpose of this study was to conduct a systematic review and meta-analysis of the effects of NO 3 − supplementation on single and repeated bouts of short-duration large muscle mass exercise in healthy humans. A secondary purpose was to conduct sub-analyses to evaluate the influence of the NO 3 − supplementation dose and duration, participant sex, exercise type (single vs. repeated sprints), exercise duration, and plasma NO 3 − and NO 2 − concentrations ([NO 3 − ] and [NO 2 − ], respectively) to further refine understanding of the experimental conditions in which NO 3 − supplementation is more likely to enhance single and repeated bouts of short-duration large muscle mass exercise.

Materials and Methods
This systematic review and meta-analysis was reported according to Preferred Reporting items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [49]. The study protocol was registered with the Center for Open Science organisation (registration number: 10.17605/OSF.IO/JSGKM).

Inclusion and Exclusion Criteria
Three researchers (N.S.A., S.J.B., and T.C.) agreed on the inclusion and exclusion criteria. These were based on a Population, Intervention, Comparator, Outcome, Study design (PICOS) methodology (see Online Supplementary Material). Briefly, studies were included if they met the following criteria: (1) participants were healthy adults ≥16 years old; (2) they administered oral inorganic NO 3 − supplements such as beetroot juice or sodium/potassium NO 3 − salts and provided information about the dose, frequency, and duration of supplementation; (3) they included exercise that recruited a large muscle mass such as running, cycling, and kayaking; (4) the exercise test included ≥1 high-intensity effort (≥ . VO 2peak ), with each effort ≤60 s; (5) they measured performance as completion time, total distance covered, maximal or mean power output, total work performed, or maximal number of repetitions. Studies were excluded if participants were <16 years old or had a chronic medical condition; NO 3 − was administered with another dietary supplement; there was insufficient information about the dose, frequency, and duration of supplementation; exercise was submaximal (≤ . VO 2max ) or if any single effort was ≥60 s; and if exercise was performed in hypoxic or hot conditions.

Search Strategy
We searched Medline and SPORTdiscus databases for English language papers from inception to January 2023. Our search strategy was based on our PICOS methodology and the full search terms for both databases are presented in the Online Supplementary Material. The reference lists of eligible full text articles were also searched to identify any other potential studies for inclusion.

Study Selection
The search results were downloaded into Rayyan software, a web tool for screening abstracts [50]. After removing duplicates, two researchers (N.S.A. and S.N.R.) independently screened titles and abstracts for inclusion. Full texts of studies deemed eligible were retrieved and compared against the predefined PICOS criteria. Where there was disagreement on whether a study should be included or excluded from the systematic review and meta-analysis, this was discussed with, and resolved by, a third researcher (S.J.B.). The study selection process is summarised in Figure 1.

Data Extraction
Data were extracted into a Microsoft Excel Spreadsheet by one researcher (N.S.A.) and substantiated by a second researcher (S.N.R.). The spreadsheet was designed and trialled by three authors (N.S.A., T.C., and S.J.B.) and refined prior to extraction. The following data and information were extracted: study design, sample size, participant characteristics (age, training status, V O2peak/max), supplementation protocol (type, dose, frequency, duration, timing of last dose relative to exercise onset, total exposure, placebo, and washout period between trials), exercise protocol (mode, intensity, duration, recovery between bouts, and number of repetitions), and mean ± SD of relevant outcomes, including the mean of all peak power outputs (PP), PP during the first sprint (PPFirst), PP during the last sprint (PPLast), time to reach PP (PPTime), mean power output from all repetitions (MP), MP during the first sprint (MPFirst), MP during the last sprint (MPLast), minimum power (PMin), total work performed in repeated cycling efforts (TWD), and total distance covered in the Yo-Yo IR1 running test (TDC). When standard error of the mean (SEM) was reported, SD was calculated as SD = SEM × √n, where n represents the sample size. Authors of studies included in the meta-analysis were contacted to retrieve individual participants' data for the calculation of pooled SD and correlation coefficient. For 15 studies, data for individual participants were provided [35][36][37][38]41,[51][52][53][54][55][56][57][58][59][60]. The correlation coefficient (Corr) was imputed for the studies with available individual participant data using the following formula:

Data Extraction
Data were extracted into a Microsoft Excel Spreadsheet by one researcher (N.S.A.) and substantiated by a second researcher (S.N.R.). The spreadsheet was designed and trialled by three authors (N.S.A., T.C., and S.J.B.) and refined prior to extraction. The following data and information were extracted: study design, sample size, participant characteristics (age, training status, . VO 2peak/max ), supplementation protocol (type, dose, frequency, duration, timing of last dose relative to exercise onset, total exposure, placebo, and washout period between trials), exercise protocol (mode, intensity, duration, recovery between bouts, and number of repetitions), and mean ± SD of relevant outcomes, including the mean of all peak power outputs (PP), PP during the first sprint (PP First ), PP during the last sprint (PP Last ), time to reach PP (PP Time ), mean power output from all repetitions (MP), MP during the first sprint (MP First ), MP during the last sprint (MP Last ), minimum power (P Min ), total work performed in repeated cycling efforts (TWD), and total distance covered in the Yo-Yo IR1 running test (TDC). When standard error of the mean (SEM) was reported, SD was calculated as SD = SEM × √ n, where n represents the sample size. Authors of studies included in the meta-analysis were contacted to retrieve individual participants' data for the calculation of pooled SD and correlation coefficient. For 15 studies, data for individual participants were provided [35][36][37][38]41,[51][52][53][54][55][56][57][58][59][60]. The correlation coefficient (Corr) was imputed for the studies with available individual participant data using the following formula: where: Antioxidants 2023, 12, 1194 5 of 20 Corr = correlation, SD E = standard deviation for the NO 3 − trial, SD C = standard deviation for the placebo trial, SD diff = the difference between the standard deviation for the NO 3 − trial and standard deviation for the placebo trial. Subsequently, the standard error of the SMD (SE(SMD)) was calculated using the formula: where: SE(SMD) = the standard error for the standardised mean difference, n = sample size, and Corr = correlation coefficient.

Quality Assessment
Risk of bias of included studies was assessed using the Revised Cochrane Collaboration risk of bias tool (ROB2) for crossover trials [68], which assesses studies based on five specific domains: (1) randomisation process; (2) deviations from the intended outcome; (3) missing outcome data; (4) measurement of the outcome; and (5) selection of the reported results. This was performed on the Cochrane excel tool available at https://www.riskofbias.info (accessed on 31 January 2022), which allows an entry for each domain in a risk of bias table rated as "low risk", "some concerns", or "high risk". Two researchers (N.S.A., and A.A.) independently evaluated the risk of bias for each study and any discrepancies were resolved through discussion. As previously recommended [69], funnel plot asymmetry was visually inspected to assess publication bias for meta-analyses that included ≥10 studies.

Statistical Analysis
Quantitative synthesis was only performed if ≥2 studies measured the same outcome. The meta-analysis was conducted using RevMan 5.4v [70]. A separate meta-analysis was performed for each of the following continuous outcomes: PP, PP First , PP Last , MP, MP First , MP Last , PP Time , TWD, and TDC. Data are presented as forest plots with 95% confidence intervals. Due to significant between-study heterogeneity, effect sizes were calculated with an inverse variance random-effects model using the DerSimonian-Laird method [71]. Effect sizes were interpreted according to Cohen's guidelines where an SMD of 0.2, 0.5, and 0.8, respectively, reflect small, medium, and large effects [72]. Heterogeneity was assessed using the Chi 2 and I 2 statistics. A value of p ≤ 0.10 on the Chi 2 test was considered significant. The I 2 was interpreted as follows: <25%, low risk; 25-75%, moderate risk; and >75% high risk [69]. Additionally, forest plots were visually inspected to check for observable differences in study results. A sensitivity analysis was conducted by using a correlation coefficient of 0.5 for all studies [73], removing studies that had a high risk of bias for at least one domain, and those with elite endurance athletes, as previous studies have reported that dietary NO 3 − supplementation is less effective in this population [60,63]. For sub-group analysis, the influence of the NO 3 − supplementation dose (<8 mmol vs. ≥8 mmol) and duration (single day vs. multiple days supplementation), exercise type (single vs. repeated sprints), and exercise duration (≤15 s vs. >15 s-≤30 s) were assessed. Due to the low number of studies that measured plasma [NO 3 − ] and [NO 2 − ] and included female participants, a sub-group analysis on the influence of plasma [NO 3 − ] and [NO 2 − ] and biological sex could not be performed. Studies recruiting well-trained endurance athletes were omitted from sub-group analyses on the basis that this population group does not exhibit an ergogenic effect after NO 3 − supplementation [60,63]. Statistical significance was accepted at p < 0.05.

Discussion
The principal observations of this systematic review and meta-analyses are that, compared to a placebo condition, NO 3 − supplementation lowered PP Time without impacting PP, increased MP and MP First , and increased TDC in the Yo-Yo IR1 test. The improvement in MP after NO 3 − supplementation was more likely to occur when NO 3 − was administered for multiple days at a dose ≥ 8 mmol as opposed to an acute serving of <8 mmol during a single bout rather than repeated bouts of high-intensity exercise, and when the high-intensity exercise duration was >15 s-≤30 s versus ≤15 s. The sub-group analysis also revealed that NO 3 − supplementation was more likely to improve TWD in a highintensity repeated bout protocol when NO 3 − was administered at a dose ≥ 8 mmol and was supplemented for multiple days as opposed to an acute serving or a dose < 8 mmol. These observations improve our understanding of the effects of NO 3 − supplementation on single and repeated bouts of short-duration, high-intensity, large muscle mass exercise, and reveal two apparently distinct and supplementation-strategy-dependent effects of dietary NO 3 − on high-intensity exercise performance. Firstly, NO 3 − supplementation appears to improve PP Time and MP First , with the improvements in these variables not necessarily requiring multiple-day supplementation with ≥8 mmol NO 3 − , as such effects appear to be achievable after acute supplementation with~6 mmol NO 3 − . Secondly, TWD in a repeated sprint protocol was more likely to be improved when NO 3 − was administered at a dose ≥ 8 mmol, and was supplemented for multiple days, consistent with the NO 3 − supplementation regime administered in the studies assessing TDC in the Yo-Yo IR1 test, all of which reported improved performance. Therefore, it appears that a single bout of high-intensity exercise can be enhanced by acute NO 3 − supplementation, with high-intensity intermittent exercise performance more likely to improve after multiple day supplementation, with a NO 3 − dose ≥8 mmol. These findings may have implications for future study design and for improving performance in athletes participating in sports that require high-intensity bouts of exercise.
Although there are some examples of enhanced PP after NO 3 − supplementation [35,52,54,[57][58][59]67], the current meta-analysis indicates that most previous studies did not report improved PP, PP First , or PP Last after NO 3 − supplementation [36,39,41,53,56,60,64,66]. However, whilst PP was not altered, PP Time was lowered after NO 3 − supplementation with all four studies assessing this variable observing a lower PP Time after NO 3 − supplementation [35,36,54,57], with three of these studies administering an acute NO 3 − dose of 6 mmol [35,54,57]. This observation is compatible with an increase in muscle contractile velocity, which would be expected to contribute to lower PP Time after acute NO 3 − supplementation [33,34]. With regard to MP variables, MP and MP First , but not MP Last , were improved after NO 3 − supplementation. When the improvement in MP after NO 3 − supplementation was explored further, MP was improved after NO 3 − supplementation when doses ≥ 8 mmol were administered [41,64,65], when multiple day supplementation protocols were adopted [41,64,65], and when a single sprint >15 s-≤30 s was performed [35,54,58,59]. The improvements in PP Time and MP First were exhibited after acute supplementation with~6 mmol NO 3 − [35,39,54,57]. All four studies assessing the effect of NO 3 − supplementation on TDC in the Yo-Yo IR1 test revealed a greater TDC after NO 3 − supplementation [37,38,55,62]. While TWD during high-intensity intermittent exercise was not improved after NO 3 − supplementation, the sub-group analysis revealed that TWD was increased when the NO 3 − dose was ≥8 mmol compared to <8 mmol [51,65], and with multiple-day supplementation compared to acute supplementation [51,65]. Importantly, the four studies reporting improved TDC in the Yo-Yo IR1 test all adopted a multiple-day supplementation protocol with a NO 3 − dose of >8 mmol [37,38,55,62]. Therefore, it appears that a multiple-day supplemental protocol with a NO 3 − dose of >8 mmol is important to elicit an ergogenic effect on repeated bouts of high-intensity exercise after NO 3 − supplementation but that performance in single sprints (lower PP Time and higher MP) can be enhanced after acute ingestion of~6 mmol NO 3 − . The ergogenic effect of NO 3 − supplementation has been attributed to its stepwise reduction to NO 2 − and the subsequent reduction of NO 2 − to NO [2,5]. It is now recognised that~25% of ingested NO 3 − is extracted from the circulation by the salivary glands [76] via the NO 3 − /H + cotransporter, sialin [77]. NO 3 − is subsequently concentrated within salivary glands [78] with excreted salivary NO 3 − undergoing reduction to NO 2 − by certain species of the oral micobiome [79][80][81]. NO 2 − -rich saliva is then swallowed and subsequently reduced to NO and various reactive nitrogen intermediates, including S-nitrosothiols (RSNO) within the stomach [2,78] − ] in skeletal muscle than blood [88,89]. The NO 3 − transporter, sialin, has been identified in skeletal muscle [89,90] which, together with chloride channel 1 [90], facilitate the concentration of NO 3 − within skeletal muscle. Therefore, a portion of the increased circulating blood NO 3 − after NO 3 − supplementation, which is not extracted by the kidney for clearance in the urine or absorbed by the salivary glands for subsequent oral reduction to NO 2 − , can be accrued in skeletal muscle. Indeed, skeletal muscle [NO 3 − ] and [NO 2 − ] are increased following NO 3 − supplementation with duration-dependent increases at least up to 7 days of supplementation [88]. In addition to its role as a NO 2 − reductase [91], xanthine oxidoreductase (XOR) can function as a NO 3 − reductase to increase NO 2 − synthesis [92] and is present in skeletal muscle [89,90]. It has been reported that the increase in skeletal muscle [NO 2 − ] after NO 3 − administration is enhanced by exercise and, as muscle pH is lowered, with both NO 3 − reduction to NO 2 − and NO 2 − reduction to NO abolished after XOR inhibition [93]. It is, therefore, possible that increased XOR activity during exercise, particularly high-intensity exercise [94], could contribute to enhanced muscle NO 3 − and NO 2 − reduction in such settings. Indeed, the increase in skeletal muscle [NO 3 − ] after NO 3 − supplementation is lowered following the completion of exhaustive cycling exercise [89] and maximal knee extensor contractions [87], suggesting that this elevated muscle NO 3 − pool is utilised as a substrate for sequential reduction to NO 2 − and then NO. There is also a positive arterial-venous difference in plasma [NO 3 − ] and [NO 2 − ] across contracting skeletal muscles after NO 3 − supplementation [95]. Since NO 2 − reduction to NO is augmented in hypoxia and acidosis [26][27][28], and given that such conditions develop within the muscle microvasculature during exercise in an intensity-dependent manner [31], elevating circulating plasma [NO 2 − ] is likely to increase NO synthesis in the muscle microvasculature during high-intensity exercise. Based on the existing evidence, NO 3 − and NO 2 − can be increased systemically and within skeletal muscle following dietary NO 3 − supplementation with the potential to enhance NO synthesis, particularly during the hypoxic and acidic conditions that develop during high-intensity exercise, which might underpin the improvements in high-intensity exercise performance variables reported in this manuscript.
The improvements in PP Time and MP First during an all-out sprint after NO 3 − supplementation are likely mediated by mechanisms intrinsic to the myocytes. The initial stages of a short-duration all-out sprint, during which PP Time will be determined, will involve maximal recruitment of, and proportion contribution to force production from, type II skeletal muscle fibres [96,97]. Previous research has indicated that 7 days NO 3 − supplementation can increase calcium (Ca 2+ ) handling proteins and evoke force production in type II skeletal muscle, but not slow-twitch (type I) skeletal muscle, in mice [18]. However, three [35,54,57] of the four [35,36,54,57] studies reporting improved PP Time , and six [39,54,56,57,64,67] of the eleven [36,39,41,54,[56][57][58]60,61,64,67] studies reporting improved MP First after NO 3 − supplementation administered NO 3 − acutely, and it has been reported that increased evoked muscle force production can occur independently of changes in Ca 2+ handling proteins in human skeletal muscle [98]. Therefore, the improvements in PP Time and MP First after NO 3 − supplementation are likely to be underpinned by NO-cyclic guanosine monophosphate (cGMP)-mediated signalling and/or post-translational modification of protein thiols [99].
In contrast to the NO 3 − supplementation regime required to improve PP Time and MP First , TWD during high-intensity intermittent exercise was improved after NO 3 − supple-  [88]. Indeed, when mouse single myocytes were acutely exposed to increased NO 2 − , contractile function and Ca 2+ handling were not altered in the earlier stages of a fatigue-inducing contraction protocol, whereas time to task failure was extended as a result of better maintenance of myocyte contractility, Ca 2+ sensitivity, and Ca 2+ pumping towards the latter stages of the protocol [100]. In human skeletal muscle, greater potential for improved muscle contractile responses during a fatigue-inducing 60 maximum voluntary contraction protocol has been reported during the initial contractions after acute NO 3 − ingestion [87] and following completion of the fatiguing protocol after multiple-day NO 3 − supplementation [101]. Skeletal muscle [NO 3 − ] and [NO 2 − ] increase in a duration-dependent manner following NO 3 − supplementation [88], and muscle [NO 3 − ] declines during sustained high-intensity exercise [87,89] and is correlated with improved muscle force production [87]. Therefore, multiple-day NO 3 − supplementation with a NO 3 − dose exceeding 8 mmol may be more effective at improving MP during a single 15-30 s bout of high-intensity exercise or at improving TWD or TDC during high-intensity intermittent exercise by eliciting greater increases in muscle [NO 3 − ] and [NO 2 − ] to support greater NO 3 − reduction and NO generating potential during these high-intensity exercise settings. As such, NO 3 − may impact skeletal muscle contractile function in a supplementation-strategy-dependent manner that may be mediated by different muscle exposures to NO 3 − and NO 2 − . Although the findings of the current study may have implications for improving NO 3 − supplementation strategies to bolster performance in different types of high-intensity exercise, there are several limitations of, and experimental considerations from, the studies included in this systematic review and meta-analysis. Firstly, the SMD was typically small across all variables that did exhibit an ergogenic effect after NO 3 − supplementation, which underscores the importance of assessing the translational potential of these findings to improve in-competition performance in sports where performance outcomes are dictated by the capability to perform high-intensity exercise. Moreover, the meta-analysis conducted on PP Time and MP exhibited high heterogeneity, indicating a substantial variation in the results of the included studies. Since a limited number of studies assessed plasma [NO 3 − ] and [NO 2 − ] and included female participants, not all planned sub-analyses could be completed. There was also limited assessment of the physiological mechanisms for any improvement in high-intensity exercise performance in the studies included in the current systematic review and meta-analyses. Therefore, further research is required to resolve the putative mechanisms for improved performance during single and repeated bouts of short duration high-intensity exercise and the extent to which such mechanisms are influenced by acute and multiple-day NO 3 − ingestion and mediated by plasma and muscle [NO 3 − ] and [NO 2 − ] and different population groups

Conclusions
The current study conducted a systematic review and completed several meta analyses to evaluate the effect of dietary NO 3 − supplementation of different aspects of high-intensity exercise performance, with sub-analyses conducted to provide wider contextual insight. It was observed that NO 3 − supplementation lowered PP Time , increased MP and MP First , and increased TDC in the Yo-Yo IR1 test, supporting the ergogenic potential of dietary NO 3 − supplementation for some aspects of high-intensity exercise performance. Sub-group analyses revealed that MP was more likely to be improved during a single >15 s-≤30 s versus ≤15 s bout rather than repeated bouts of high-intensity exercise, and that MP, TWD, and TDC were more likely to be improved after multiple-day supplementation with a daily NO 3 − dose ≥8 mmol compared to acute ingestion of <8 mmol NO 3 − . These findings improve our understanding of the ergogenic potential of dietary NO 3 − supplementation for high-intensity exercise and can help inform NO 3 − supplementation strategies to improve high-intensity exercise performance.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12061194/s1: Search strategy; Table S1: Population, Intervention, Comparator, Outcome, Study design (PICOS) framework for study eligibility; Figure S1: Risk of bias summary for individual for crossover trials; Figure S2: Funnel plot evaluating publication bias of trials assessing mean peak power output (n = 12); Figure S3: Funnel plot evaluating publication bias of trials assessing mean of the mean power output (n = 12); Figure S4: Funnel plot evaluating publication bias of trials assessing mean power output during the first sprint (n = 10); Figure S5: Funnel plot evaluating publication bias of trials assessing mean power output during the last sprint (n = 10); Figure S6: Forrest plot for mean peak power output (a), peak power during the first sprint (b), and peak power during the last sprint (c) in the nitrate and placebo trials; Figure S7: Forrest plot for mean power output sub-group analyse; low nitrate dose <8 mmol compared to high nitrate dose ≥8 mmol (a), single day nitrate supplementation compared multiple days nitrate supplementation (b), single sprint compared to repeated sprints (c), exercise duration ≤ 15 s compared to exercise duration >15 s-≤30 s (d); Figure S8: Forrest plot for total work done in the nitrate and placebo trials.