Nitrate is an inorganic anion present in the environment in various foods, especially vegetables such as celery, beetroot, lettuce and spinach [1
]. After its consumption, nitrate circulates through the plasma, with an average half-life of 5 h. After it is absorbed in the blood, about 25% returns to the salivary glands, through an active transport, and concentrates in the saliva, with the rest being excreted by the kidneys. Nitrate concentrated in saliva is converted to nitrite by facultative commensal bacteria, which reside in crypts on the surface of the tongue. After that, this nitrite can be converted to nitric oxide in the stomach, due to its acidity becoming available to the organism [1
The daily doses of 4.1 mmol to 16.8 mmol (approximately 250 mg to 1 g) of nitrate, consumed from 2 to 15 days, increase nitrite levels in the blood [1
]. A review article showed that the typical averages used in studies range from 5 mmol to 9 mmol (approximately 300 mg to 550 mg) [1
]. Nitrate is consumed usually between 1.5 h and 3 h before exercise, in a single dose up to five times per day [6
Studies have shown that nitrate supplementation promotes vasodilatation, increases blood flow to the muscle, favoring the uptake of nutrients in the skeletal muscle and muscle contraction, attenuating the release of excess calcium, and subsequently reducing the ATP production cost [12
]. Larsen et al. [14
] showed that 0.1 mmol/kg/day of sodium nitrate supplementation in isolated skeletal muscle mitochondria promotes higher respiratory control than mitochondria from non-supplemented controls. Lansley et al. [15
] showed that nitrate supplementation (6.2 mmol of nitrate in beetroot juice) for six days decreased 7% the oxygen amount required for constant rate moderate work and 15% in severe intensity running. Therefore, the low cost of oxygen in exercises with submaximal intensity, the greater mitochondrial efficiency, and physiological responses of fast twitch fibers (type II fibers), which can reduce NO3
, improving local perfusion, fatigue resistance, and muscle fiber contraction could improve performance of runners in sprint races [11
Some investigations analyzed the performance effects of nitrate supplementation on runners. The meta-analysis from Hoon et al. [18
] demonstrated that the nitrate supplementation had a minor benefit on time-trial (TT) performance and graded exercise tests in trained participants. Jones (2014) also showed that greater effects occurred when nitrate was ingested chronically and exercise was less than 30 min, such as short bouts or sprints. de Castro et al. [19
] verified the effects of chronic nitrate supplementation on 10-km running performance in recreational runners and they administered 420 mL of beetroot juice for three days, and on the days of the assessments, the ingestion occurred 2 h before the test. The authors observed lower time to complete the first half of the test (5 km) compared to placebo, however, there were no statistically significant difference in the performance of the 10-km run. Thus, it seems that trained participants usually present minor responses to nitrate supplementation [20
]. Therefore, there was no investigation of the ergogenicity of nitrate ingestion during a training program. One may hypothesize that the chronic ingestion of nitrate may enhance the responses to training, and, thereby, further improve performance.
Therefore, the purpose of the present study was to verify the effects of inorganic nitrate supplementation combined with a periodized running program on two different outcomes; the primary was 10-km running TT performance and the power developed during a Wingate test, and the second was the [La−] in the mentioned tests in recreationally trained runners.
presents the mean and standard deviation values for age, body weight, height, fat-free body mass, and % fat at baseline in the placebo and nitrate groups. There were no statistically significant differences between groups at baseline for any variables investigated, reassuring the efficiency of the randomization protocol.
shows the time to complete the five 10-km running TTs and the power developed in the 60-s Wingate tests for placebo and nitrate groups.
We observed a main effect of time (F = 21.302, p < 0.001, ES = 0.68) and significant group × time interaction (F = 13.387, p < 0.001) for the time to complete the TT. Post-hoc analysis revealed that time decreased significantly in all timepoints compared to baseline, and, more importantly, that the nitrate group was faster (−3.2 min) in week 2 than week 1, whereas there was no difference in the placebo group. There were no significant differences in the baseline values comparing both groups.
For maximum power developed during the Wingate test, there was a main effect of time (F = 12.641, p < 0.001, ES = 0.47), but no interaction (F = 1.869, p = 0.129) or significant difference between group were observed (F = 3.028, p = 0.104). There was, however, a significant group × time interaction (F = 2.934, p = 0.028) for average power, but the post-hoc analysis did not identify significant difference between groups. There were no significant differences in the baseline values comparing both groups.
presents the differences in the lactate concentration in the 10-km running test and the 60-s Wingate test weekly between placebo and nitrate groups.
For lactate during 10-km running, there was a significant group × time interaction (F = 5.943, p < 0.001) with lower [La−] in the nitrate group (F = 4.942, p = 0.043) compared to placebo at week 2 (p = 0.032), week 3 (p = 0.002), and week 4 (p = 0.003). Post hoc analysis also showed higher [La−] in the placebo group after week 3 compared to week 1 (p = 0.006) and week 5 in relation to baseline (p = 0.044) and week 1 (p = 0.011).
During the Wingate test, there was a main effect of time (F =16.891, p < 0.001, ES = 0.55), but no group × time interaction (F = 2.368, p = 0.064).
There were no significant differences in the baseline values comparing both groups for these two variables.
Several studies have examined the acute and short-term chronic impact of nitrate supplementation in different exercise modalities. To our best knowledge, this study is the first to analyze the effects of a four-week periodized training program supplemented with daily nitrate ingestion on a 10-km running TT and power developed during a 60-s maximal cycling test in recreational runners. The main finding of this study is that nitrate supplementation improved performance within 7 days and induced an additional improvement at 14 days in a 30-day protocol. This performance improvement was concomitant with a lowering in blood [La−] during exercise within 14 days. Nitrates did not, however, change the performance indices associated with more “anaerobic” performance.
Plasma nitrite levels are a highly sensitive marker of NO bioavailability. Several researchers showed that nitrate supplementation increases plasma nitrite levels at low doses (4.1 mmol) and high doses (16.8 mmol) [6
], both in acute supplementation and in chronic supplementation [8
] proving that nitrate supplementation increases nitric oxide concentration. Thus, a supplementation of 750 mg of nitrate (approximately 12 mmol), as used in this study, is likely to increase the concentration of nitric oxide. The current results of enhanced performance during an approximately 58 min long exercise add to the literature. In fact, a systematic review published by Dominguez et al. [24
] showed that nitrate supplementation improves cardiorespiratory endurance in exercises shorter than 30 min. Another meta-analysis showed that nitrate supplementation, despite presenting smalls effects, had significantly greater effect size when compared to a placebo control in a time-to-exhaustion protocol, but in time-trial performance, the small effects of the nitrate supplementation were not significant [25
]. Usually, studies with exercises over 30 min are performed with athletes. Previous studies have shown that men with VO2máx
> 60 mL·kg−1
and women with VO2máx
> 50 mL·kg−1
do not present significant enhancements with nitrate supplementation because these participants have a high level of fitness or have high levels of nitrate or nitrite in the blood, with a lower response with the use of this supplement [20
]. Since our study was performed with recreational runners, the lower fitness may explain the positive effects reported here.
Possibly the greatest aspect of this study is the weekly analysis of the impact of nitrate supplementation over the course of a four-week periodized training program. Previous investigations have used acute (2–2.5 h prior to exercise) or short-term (1 to 15 days) chronic nitrate supplementation [27
]. Investigations of longer supplementation periods with weekly performance analysis are scarce. Wylie et al. [28
] used doses of 3 mmol and 6 mmol of nitrate over 30 days, with analyses at 7, 28, and 30 days, and showed that the largest dose can reduce submaximal exercise O2
cost. The decrease in the cost of O2
might be associated with greater running economy and consequently improved performance over long distances. Those results would help explain the data in our study [29
]. In addition, our data show a performance improvement in the 7th day, results similar to those found by Wylie et al. [28
], but also demonstrates an additional improvement in the 14th day. These data bridge a gap in the literature since the performance improvement results with chronic nitrate supplementation were reported to occur between 1 and 15 days or after 28 to 30 days of supplementation, with no result being registered between 15 and 28 days of supplementation. This helps refine current supplementation recommendation for moderate-training runners.
Previous studies have shown that running long distances increase blood [La−
], decreasing performance, although lactate is not the cause of fatigue per se [30
]. A study from our research group showed that high blood [La−
] are associated with impaired performance in a 10-km running time trial, corroborating this information [21
]. Nitrate supplementation did not change blood [La−
] compared to placebo in well-trained 10-km runners as shown in a similar study [23
]. However, highly trained participants or athletes compared to recreational runners presented lower [La−
] with nitrate supplementation given in an acute manner (~12.5 mmol acute supplementation), which is different from our study, since it was made with recreational runners with nitrate given in a chronic way, which can explain these conflicting results. Our results then suggest that nitrate could have shifted the energy supply from an anaerobic source to an oxidative supply, as stated by Wylie et al. [28
]. These results indicate that the benefits of nitrate supplementation on blood [La−
] in long-distance running or high-intensity exercises are dependent on the supplementation period/protocol (acute or chronic) and of the level of physical fitness of the subject.
We also examined changes in Wingate test performance since runners have been shown to enhance their running economy and endurance performance with high-intensity training [33
]. Our study did not show significant effects on maximal power on Wingate Test in the recreational runners. Previous studies show contradictory results. Dominguez et al. [35
] presented performance improvements in maximum power on a 30-s Wingate test with acute nitrate supplementation (~5.6 mmol) during the first 15 s. Other studies show no performance improvements in maximum power 30-s Wingate tests with six days of nitrate supplementation (~800 mg per day) [36
]. Future studies are needed to better understand the mechanisms involved with anaerobic power and nitrate supplementation.
A possible explanation of this result is that the participants in this study selected aerobic exercise as their activity of choice, with greater adaptation on slow-twitch fibers and smaller adaptation in fast-twitch fibers, probably not enough to be detected in the Wingate test. Furthermore, nitrate supplementation dosage, period of supplementation, subject fitness levels, and time of the Wingate test protocol (60 s vs. 30 s, which is usually used), might have interfered with our results.
The difference in fat free body mass (p = 0.071) between groups is another important element, although it was not significant.
Limitations of Study
This study presents some limitations. We acknowledge that the number of participants was low and may not be fully representative of the runner population. However, the selection of participants was rigorous. The inclusion and exclusion criteria limited the number of participants, mainly due to the use of any supplements or ergogenic substances during the experimental protocol. Most runners did not want to stop using other supplements during the study.
Control of supplement intake and training was also a limitation of the study. Daily contact was made with everyone, but this contact was sometimes only by text messages, thus some training and supplement intake was not personally verified.
We did not perform the nitrite concentration analysis in the plasma. This analysis would give us further important information that would facilitate the understanding of our data.