Influence of Nitrate Supplementation on Endurance Cyclic Sports Performance: A Systematic Review

This systematic review focused on the effect of nitrate supplementation on endurance cyclic sports performance. This review followed the PICOS question model for framing the research question and defining the inclusion criteria [19]: P (population): “endurance and healthy trained athletes between 18 and 50 years old”; I (intervention): “supplementation with nitrate”; C (comparison): “same conditions with placebo or control group”; O (outcomes): “performance measures”; S (study design): “double- or single-blind design and randomized parallel or crossed.”

A structured search was carried out following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines [20] in the SCOPUS, Web of Science (WOS), Sport Discus, and Medline (PubMed) databases. The search included original articles with randomized controlled crossover or parallel designs in which the intake of nitrate administered before and/or after the exercise was compared with an identical placebo situation. Due to the increasing number of investigations over the last 10 years interested in using nitrate as a supplement in athletes, the search was bounded from 1 January 2010 to 30 November 2019. Search terms were established matching the Medical Subject Headings (MeSH), as follows: “nitrate,” “nitric oxide,” “beetroot juice,” “performance exercise,” and “endurance.” The connectors OR and AND were also used to filter the search.

The following inclusion criteria were applied to select studies: (I) articles depicting a well-designed experiment that included the ingestion of a dose of nitrate, or nitric oxide or beetroot juice before and/or during exercise in healthy trained athletes; (II) the study investigated endurance cyclic sports; (III) there was an identical experimental situation with or without the ingestion of a placebo in a normoxic condition; (IV) there was a double- or single-blind design and randomized parallel or crossed design; (V) crossed over studies that did not allow for a clean-up period of over 24 h were excluded; (VI) there was clear information on the administration of nitrate; (VII) nitrate was administered in the form of a beverage, gum, or pills; (VIII) at least one of the measured performance variables was changes (effort shown, maximum power, average power, time, distance, oxygen consumption, saturated oxygen, total work, or number of completed sprints among others); and (IX) the language was restricted to English. The following exclusion criteria were applied: (I) animal studies, (II) uncontrolled trials, (III) studies using non-standardized turmeric extracts or extracts of unknown nitrate content, (IV) studies performed on non-trained or injured/sick subjects, (V) studies investigating children and the elderly, and (VI) studies in non-specific sports or non-endurance sports.

The following information was extracted from the selected articles: study source (authors and year of publication), participant characteristics (level of activity or sports discipline, experience and training load described by the authors, number of participants and sex), age, VO_2max, supplementation protocol (source of nitrates and duration), nitrate dose, time from the last dose of nitrate, exercise protocol, main outcome (performance measures), and results.

The methodological quality of the articles, evaluated using McMaster’s Critical Review Form [21], ranged between 13 and 15 points, representing a minimum methodological quality of 86.6% and a maximum of 100%. Of the 27 studies, 3 achieved 13 items, representing 11.11% of the total number of studies, 3 achieved 14 items, representing the same percentage of 11.11%, and the rest of the studies (21) achieved 15 items, representing 77.78% of the total number of studies. All studies achieved a “very good” quality. No study was excluded because it did not reach the minimum quality threshold. The main deficiencies found in methodological quality were associated with items 13 and 14 of the questionnaire and comprised the report of clinical importance and one study had drop-outs. The objective of this evaluation was to determine and to compare the quality between the different study designs (Table 2).

The initial research obtained through database searches provided 4341 records. Of these records, 3102 duplicates were removed. Of the 1239 remaining records, a total of 1067 were excluded after screening the titles and abstracts for eligibility. Ultimately, 27 studies were included in this review. The PRISMA flow chart was used to summarize the systematic research and selection process (Figure 2).

A total of 323 participants were included in these 27 studies. The average sample included 12 subjects but seven studies assessed the effects of nitrate in larger group subjects (up to 26 participants). The study mode was carried out using eight subjects. All subjects in the review were defined by the authors as either trained or well-trained using the various criteria listed in Table 3. Among the 323 experimental subjects, only 39 (12.07%) were women. Moreover, female athletes only took part in 5 of the 27 studies selected, with only one study exclusively on trained women. Most of the studies were performed with populations in their 20s and 30s. Most focused on cycling, with 9 studies (33.33%) and 101 participants (31.27%), followed by combined cycling and triathlon studies, with 97 participants (30%) and 6 studies (22.2%). We also found other less represented sports, such as swimming (3), athletics (2), rowing (1), and cross-country skiing (2). For the ergogenic effect of nitrate supplementation, statistically significant results were obtained in eight (29.63%) of the studies that investigated cyclic endurance sports performance [22,23,26,33,35,44,45].

The main form of supplementation was beetroot, except for three studies that used sodium nitrate [18,23,40] and one that used potassium nitrate [42]. Most of the studies used mmol as a measuring unit to determine the value of the dose of nitrate intake. The average dose of nitrate was 8.7 mmol/day (minimum 4 mmol/day and a 19.5 mmol/day maximum). Both acute and chronic supplementation protocols were found in the reviewed studies. Of the studies, 40.74% used chronic supplementation and 51.85% used acute supplementation, applying equally high and low doses for both acute and chronic treatments, and 7.4% used both types of supplementation.

Studies on chronic treatment were performed over an average of 6 days, taking into account the fact that some treatments lasted 3 days, and others, such as those described by Balsalobre et al. [22], applied supplementation for 15 days. The last dose of nitrate before physical performance testing was not given at the same moment in all the studies. The intervals used for administering the dose before the performance test was between 1.5 and 4 h, with the most common being 2–3 h (88.89%). Only the study of Balsalobre et al. [22], which featured the longest chronic supplementation (15 days), began the incremental test until exhaustion 24 h after the last dose.

Athletes in this systematic review had a moderate-to-high level of cardiorespiratory fitness. Most of the studies determined the VO_2max of their athletes. The VO_2max values ranged from 42 mL/kg/min to 85 mL/kg/min, with the lowest values found in studies using male master athletes in swimming [43]. Other measures of performance used to determine the effects of nitrate supplementation were time, distance average, and maximum power. Moreover, except for some gastrointestinal pain, secondary effects were not reported, and all doses of supplementation were tolerated. For example, in the study by Hoon et al. [33], two athletes described mild gastrointestinal pain after consuming a nitrate-rich drink.

Most of the studies did not present a risk of bias. They were carried out using healthy and uninjured athletes via a randomized double-blind control test, leaving a sufficiently long clean-up period lasting from 4 to 7 days (except for some that did not use a crossover). Approximately 10% were done using simple blind tests. The placebo drinks, in most cases, had the same packaging, color, smell, and taste as the nitrate-rich drinks to reduce the risk of bias.

Most studies gave clear instructions to participants to refrain from caffeine and alcohol intake for at least 24 h before performance testing. Further, identical diets were used in the experiments such that diet would not influence the results. Details of nitrate-rich foods (green vegetables, beets, strawberries, grapes, and tea) and the importance of eliminating them from one’s diet during the period of experimentation were given. Most participants maintained a moderate level of training during the previous days, in addition to refraining from physical exercise during the last 24 h before testing.

In all studies, the concentrations of nitrate and plasma nitrite increased significantly (p < 0.05) after nitrate supplementation compared to the placebo. Among the 27 studies reviewed, about 29.63% provided results that were statistically significant for nitrate intake. As shown in Table 4, of the eight articles, four showed significant results for both respiratory parameters and measures of performance [22,23,43,44], seven studies give results for measures of performance [22,26,33,35,43,44,45] of which three did not influence respiratory parameters [33,35,45], and five significant studies gave results on respiratory parameters [22,23,26,43,44], of which only one did not influence the measures of performance [23].

Boorsma et al. [24] used the highest acute (13 mmol) and chronic (19.5 mmol) nitrate doses of all the studies. Nevertheless, the two experimental tests (the incremental test and 1500 m timed trial) provided no significant results. Bescós et al. [23] demonstrated a significant improvement in oxygen consumption during their tests, but in neither case did this improvement increase sporting performance. The other studies showed that nitrate supplementation did not increase VO₂ or improve performance.

This systematic review demonstrates that the ergogenic effects of nitrate supplementation on endurance cyclic sports performance occur mainly through the intake of beetroot juice. However, the consumption of beetroot juice allows for a substantial nitrate load to be ingested quickly and easily before training or completion. Beetroot juice is considered an ergogenic supplement with strong scientific evidence, according to the ABCD classification of the Australian Sports Institute (AIS) [8]. In the included investigations, a placebo drink with an insignificant nitrate content was used. In addition to nitrate, beetroot also contains several active compounds that can influence physiological responses to exercise, including betaine, antioxidants, and polyphenols [35]. The use of beetroot depleted in nitrate as a placebo allowed the effects of nitrate to be isolated from those of other potentially ergogenic components.

Other studies that applied different forms of nitrate supplementation, such as potassium nitrate or sodium nitrate, did not obtain significant results. Thus, this form of supplementation may be less effective than beetroot juice. For example, a study conducted in 2016 compared the effect of beetroot juice and sodium nitrate on performance in trained athletes and discovered that the same concentration of nitrate produced a larger reduction of oxygen uptake in the experimental group that used beet juice [3], but the study did not highlight any factors related to sports performance.

Balsalobre et al. [22], after supplementing runners with a dose of 6.5 mmol/day of nitrate for 15 days, performed an incremental test until exhaustion on a treadmill (from low to high intensity), showing significant results in time until exhaustion (S: 1269 ± 53.6 vs. PLA: 1230 ± 73.5 s), which means that the supplemented athletes performed better. The most intense part of the test was used to determine the speed associated with VO_2max.

Similarly, the incremental test with a cycloergometer performed in a study on cyclists by Bescós et al. [23] demonstrated better results with an acute dose of 11.8 mmol of nitrate compared to a placebo, but these results were not statistically significant (S: 416 ± 32 vs. PLA: 409 ± 27 s). The main difference between both studies was the supplementation protocol (beetroot juice for 15 days and sodium nitrate for a single day). As previously noted, among the 27 studies, 40.74% used a chronic dose and the other 51.85% used an acute dose, making it difficult to determine the option with the best effect. Boorsma et al. [24] and Nyakayiru et al. [40] were the only studies that investigated both types of supplementation and obtained similar results for both acute and chronic supplementation (both negative results). Similarly, McQuillan et al. [38] performed four time trials using different days of supplementation (3, 4, 6, and 7 days), and all of them gave negative results. On the other hand, Cermak conducted two studies [26,27] with triathletes and cyclists with a similar VO_2max, dose of nitrate, time between the last intake, and time trial. The only differences were the days of supplementation (6/1 day) and the total time taken for the time trial (16/60 min). Cermak et al. [26] found a significant improvement in performance, while the other study did not obtain better results for the test time. This could be due to the form of supplementation or the type of test done. Notably, the eight most significant studies [22,23,26,33,35,43,44,45] examined five different sports with different doses of supplementation (see Table 3 and Table 4); therefore, the best mode of supplementation to obtain superior performance among athletes remains uncertain.

In humans, before absorption through the stomach wall, approximately 25% of the nitrate consumed is absorbed by the salivary glands and reduced to nitrite via the action of nitrate reductase enzymes from the facultative anaerobic bacteria on the dorsal surface of the tongue. Next, this ingested nitrite is reduced to NO in the acidic environment of the stomach but a substantial amount undergoes systematic circulation as nitrite. Plasma nitrate levels increase rapidly within 30 min after supplementation, reaching the peaks of nitrate and nitrite at 1–2 h. Two to three hours later, nitrite and nitrate gradually fall, returning to the baseline after approximately 24 h. The average life of plasma nitrate in humans is approximately 5 h, with a considerable decrease 4 h after ingestion.

All studies that obtained favorable ergogenic effects from nitrate performed their experimental tests between 2 and 3 h after intake. The studies that started their tests before 2 h improved their exercise capacity, while those that started after 3 h failed to improve their exercise capacity [24,25,32,34,37,39,40]. Interestingly, only one study analyzed the effects of the supplement 24 h after intake, obtaining significant results [25].

The nitrate and nitrite levels in plasma when fasting are 20–50,000 mmol/L and 100–500 mmol/L, respectively. Additionally, estimations of the average consumption of nitrate and nitrite in Europe are 31–185 mg/day, with 100% bioavailability of nitrate in the diet [4]. Thus, the doses used in nitrate studies are 4 to 12 times greater than the typical daily intake of nitrate in Europe. Studies like that of Bescós et al. [23] used an acute intake of 10 mg per kg of body weight of sodium nitrate, representing an approximate nitrate dose of 11.8 mmol. With these values, the participants went from having similar nitrate concentrations before intake (S: 30 ± 12, PLA: 28 ± 10 µM) to having an increase in plasma nitrate levels three hours after intake in the experimental group (250 ± 80 µM), with no change observed in the placebo group (29 ± 8 µM).

Amongst the protocols used during the studies, those that decided to refrain from the use of mouthwash or chewing gum stand out. These hygienic products alter the bioavailability of nitrite by killing the oral bacteria necessary for nitrate’s bioactivation. A cross-study designed with seven healthy volunteers examined the effects of antibacterial mouthwash on the nitrite levels in saliva and plasma measured after the intake of sodium nitrate (10 mg/kg) [49]. In the control situation, the levels of nitrate and nitrite in saliva and plasma increased long after the intake of nitrate. Similarly, the use of mouthwash 15 min before the intake of sodium nitrate did not have an effect on the nitrate accumulation in saliva and plasma but prevented its conversion to nitrite in saliva and attenuated the increase in plasma nitrite. The removal of some oral bacteria with mouthwash alters the bioavailability of nitrite in plasma by limiting the bioactivation of nitrate in the mouth.

The eight studies that obtained a performance improvement via nitrate supplementation prohibited the use of mouthwash among the athletes until the experimental tests were carried out. Some tests prohibited the use of mouthwash during the entire period of supplementation [22], while others prohibited it during the last 24 h before the experimental test or from the last intake until the end of the test. On the other hand, studies such as Bescós et al. [18] and Lane et al. [34], which did not see a positive effect in the performance tests after the intake of sodium nitrate and beetroot juice, did not inform the athletes of the need to refrain from oral antibacterial products, which could have caused a smaller increase in NO.

Bescós et al. [23], through the administration of large amounts of dietetic nitrate to competitive cyclists and triathletes, determined that even with no improvement in cardiorespiratory adaptation to low and medium exercise intensity (submaximal workloads corresponding to 2.0, 2.5, 3.0, and 3.5 W·kg⁻¹ of body mass), the consumption of oxygen was much more efficiently reduced under high-intensity exercise. This highlights the importance of high-intensity exercise in the performance of cyclic sports.

The ergogenic effect of the beetroot juice was mediated by the influence of NO in the improvement of the mitochondrial oxygen efficiency, which increased vasodilation and muscular blood flow, among other factors related to exercise performance [50]. One of the effects produced by these physiological mechanisms is an improvement of the running economy, which translates into being able to run at the same pace with less effort or being able to run faster with the same energy consumption. In this systematic review, a total of 27 trials were carried out to verify the improvement of endurance through nitrate supplementation. Among them, seven trials showed significant improvement in the time of distance traveled [26,33,35,44,45]. In most of the studies that measured cyclists and triathletes with cycloergometers, different tests of 1 km up to 80 km were employed. Two studies applied a 10 km test and obtained a significant improvement in the time performance and average power when the athletes ingested the supplement within 3 h before starting the test [26,44]. It should be noted that the study by Rokkedal et al. [44] applied the same distance with a shorter time and lower oxygen consumption, which means that the athletes performed at a higher intensity.

On the other hand, Nyakayiru et al. [40] and Shannon et al. [45] did not find a significant improvement in time with the 10 km test, an acute dose, or a chronic 6-day trial. This could be due to the expected time between intake and the start of the test (≥3 h) since the ingested doses and VO_2max levels of the cyclists and triathletes in both studies were very similar. However, more studies are necessary to confirm this result.

Studies conducted with distances less than or greater than 10 km did not obtain any significant improvement in exercise capacity [18,25,27,28,31,32,37,38,39,40], except for a study where the yield was verified at 4 and 16.1 km with an acute dose of 6 mmol of beet juice and an improvement in time, and the average power was obtained [35]. This research, even using an acute intake, obtained highly beneficial results, with a time improvement of 2.7% in the 4 km race and 2.8% in the 16.1 km race. Meanwhile, acute doses of nitrate given to trained cyclists for cycling races of 80 km or more seemed to have no ergogenic effect [46]. These results suggest that, as distances become greater, the importance of intensity as a determining factor in performance decreases.

The only study on rowers showed a positive effect on the time required to complete the 2000 m test (via a remover gauge) when using a dose of 8.4 mmol of nitrate, while with a reduced dose (4.2 mmol of nitrate), this effect was not observed [33]. This study demonstrates the importance of applying a higher dose such that the NO level can have an ergogenic effect. Additionally, a 2000 m race is a test that rowers are highly accustomed to; they perform such a race monthly since it is part of the national rowing selection protocol.

The mechanism by which nitrates improve the efficiency of submaximal exercise remains contentious. It is believed that increasing NO levels after supplementation can reduce the cost of ATP production, thereby improving energy production efficiency [51]. This effect is believed to be related to a lower production cost of mitochondrial ATP [6] or muscle contraction ATP [52]. It is known that one of the costliest energy processes during skeletal muscle contractions is the pumping of calcium into the sarcoplasmic reticulum, which accounts for up to 50% of the total ATP turnover. In this way, the efficiency of oxidative phosphorylation is improved under a given relative intensity and the decomposition of phosphocreatine is reduced. In this systematic review, we found 10 investigations that evaluated whether an improvement in VO₂ is observable using incremental tests at different levels of VO_2max or maximum power (W_max). Four studies that investigated incremental tests until exhaustion obtained significant results for sports performance [22,43], except for one study that found significant results in respiratory parameters but no differences in the duration of incremental exercise between the experimental group and the placebo group [23]. This was the only study that used an acute dose, which could be one of the attributable causes. These findings show that, in athletes with a high level of training, nitrate supplementation may lead to a greater tolerance of submaximal effort, while at the same time casting doubt on the decrease in oxygen consumption. Other physiological parameters, such as VCO₂ or mean heart rate, showed no significant differences between groups; neither did the perceived exertion. Only the study by Balsalobre et al. [22] showed significant improvements in the rating of perceived exertion (RPE).

A study by Pinna et al. [43] with trained male swimmers is a clear example of improved exercise efficiency. The training frequency of swimmers ranged from 3 to 4 times/week, with a 3000–5000 m distance covered each workout. With supplementation of 5.5 mmol of nitrate for six days, an incremental test was carried out until exhaustion in the pool using an elastic cord connected to a digital dynamometer tied on a belt to the subject to study the workloads. The results showed that, through the intake of beet juice, a greater workload and lower aerobic energy consumption were achieved, together with the same VO₂.

While some studies reported that dietary supplementation with NO donors induced benefits in exercise performance, others found no positive effects. In this sense, the training status of the subjects seemed to be a determining factor. Studies with healthy untrained or moderately trained subjects showed that NO donors could improve their tolerance to aerobic and anaerobic exercise [14,15,16,17]. However, for high-level athletes, who are more adapted to training and exercise, nitrate supplementation seemed to have little to no effect, at least when the doses were equal to those used on recreational athletes [53]. Additionally, some studies found that the basal plasma nitrite levels of elite athletes were higher than those of recreational athletes, showing that plasma nitrite is directly proportional to exercise capacity [54]. Therefore, one’s aerobic level could affect the ergogenic benefits induced by dietary nitrate supplementation, and higher doses may be necessary to obtain a benefit for elite athletes. Additionally, the same doses of nitrate among elite and recreational athletes were shown to produce the same increases in plasma nitrate concentrations [55]. The ingestion of a moderate amount of inorganic nitrate might not lead to higher performance because the increase in free radicals of the nitrate–nitrite–NO pathway in well-trained subjects engaging in high-intensity exercise enhances the function of the endogenous antioxidant system of these athletes; consequently, there is a greater reduction in these free radicals. On the other hand, because they are not as heavily trained, recreationally active subjects lack adaptations and may be more exposed to oxidative damage when performing high-intensity sports. Thus, increasing the bioavailability of NO through the intake of nitrate could be a benefit for such athletes. Additionally, the benefits of nitrate supplementation may also depend on individual genetics or the effects of training on muscle oxygenation, mitochondrial function, and muscle fiber type composition. It has been suggested that nitrate supplementation may be particularly effective in improving the physiological and functional responses in type II muscle fibers [56].

It is assumed, in addition to the greater adaptation that athletes have to high-intensity sports, that some athletes will benefit more than others and that there may be responders and non-responders in a given group. In the study by Bescós et al. [18], seven subjects showed a small increase (<30%) in plasma nitrite levels after dietary supplementation with nitrate compared to placebo. These subjects were methodologically classified as “low responders” or “high responders.” Although no statistical differences were found in the performance parameters (distance and output power) between the two groups, during the 40-min time trial after treatment, there was a small reduction in VO₂ (≈1.7%), VCO₂ (≈3.4%), and the relationship between VO₂ and power (≈1.1%) in the group of high responders. Additionally, blood lactate concentrations showed a tendency to decrease at 3 min after exercise (p = 0.076) in most responding patients. Similarly, even without being able to demonstrate a significant effect in the 1500 m race, two runners responded positively to acute supplementation of 19.5 mmol nitrate and chronic supplementation of 13 mmol of nitrate over 8 days [24]. These subjects improved their results by 5.8 and 5.0 s through acute intake and by 7.0 and 0.5 s through chronic intake. Additionally, in a submaximal incremental test among trained triathletes, an improvement in distance and maintenance was observed (albeit without obtaining significant results), meaning that “the need to individually analyze the positive or negative response to this supplementation in the case of athletes trained may be the best option” [30].