Nitrate Supplementation Combined with a Running Training Program Improved Time-Trial Performance in Recreationally Trained Runners

to verify the e ects of inorganic nitrate combined to a short training program on 10-km running time-trial (TT) performance, maximum and average power on a Wingate test, and lactate concentration ([La ]) in recreational runners. Sixteen healthy participants were divided randomly into two groups: Nitrate (n=8) and placebo (n=8). The experimental group ingested 750 mg/day (~12 mmol) of nitrate plus 5 g of resistant starch, and the control group ingested 6 g of resistant starch,for 30 days.All variables were assessed at baseline and weekly over 30 days. Training took place 3x/week. The time on a 10-km TT decreased signi cantly (p<0.001) in all timepoints compared to baseline in both groups, but only the nitrate group was faster in week 2 compared to 1. There was a signi cant group time interaction (p<0.001) with lower [La] in the nitrate

were assessed at baseline and weekly over 30 days. Training took place 3x/week. The time on a 10-km TT decreased signi cantly (p<0.001) in all timepoints compared to baseline in both groups, but only the nitrate group was faster in week 2 compared to 1. There was a signi cant group time interaction (p<0.001) with lower [La] in the nitrate group at week 2 (p=0.032), week 3 (p=0.002), and week 4 (p=0.003). There was a signi cant group time interaction (p=0.028) for Wingate average power and a main e ect of time for maximum power (p<0.001) and [La ] for the 60-s Wingate test. In conclusion, nitrate ingestion during a four-week running program improved 10-km TT performance and kept blood [La ] steady when compared to placebo in recreational runners. Keywords:sport nutrition; endurance training; performance; nitrate 1. Introduction Nitrate is an inorganic anion present in the environment in various foods, especially vegetables such as celery, beetroot, lettuce and spinach [1,2]. 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 Sports2019,7, 120; doi:10.3390 /sports7050120 /journal/sports

Sports2019,7, 120 2 of 10 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 [1,3–5]. 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 [1,6–8]. 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 ve times per day [6,7,9–11]. Studies have shown that nitrate supplementation promotes vasodilatation, increases blood ow 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 [12,13]. 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 e ciency, and physiological responses of fast twitch bers (type II bers), which can reduce NO3to NO2, improving local perfusion, fatigue resistance, and muscle ber contraction could improve performance of runners in sprint races [11,16,17]. Some investigations analyzed the performance e ects of nitrate supplementation on runners. The meta-analysis from Hoon et al. [18] demonstrated that the nitrate supplementation had a minor bene t on time-trial (TT) performance and graded exercise tests in trained participants. Jones (2014) also showed that greater e ects occurred when nitrate was ingested chronically and exercise was less than 30 min, such as short bouts or sprints. de

nitrate supplementation on runners. The meta-analysis from Hoon et al. [18] demonstrated that the nitrate supplementation had a minor bene t on time-trial (TT) performance and graded exercise tests in trained participants. Jones (2014) also showed that greater e ects occurred when nitrate was ingested chronically and exercise was less than 30 min, such as short bouts or sprints. de Castro et al. [19] veri ed the e ects 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 rst half of the test (5 km) compared to placebo, however, there were no statistically signi cant di erence 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 e ects of inorganic nitrate supplementation combined with a periodized running program on two di erent 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. 2. Material and Methods 2.1. Experimental Approach to the Problem This study was a randomized trial, double-blind design. Before any intervention, all participants signed the informed consent, and the study was approved by the Research Ethics Committee. Then, the participants were divided randomly into a nitrate or placebo group. Participants performed the same running training protocol during the study (described below). The participants completed a 60-s Wingate Test and a 10-km running TT at baseline and weekly during the training program. The Wingate test and the TT were conducted at the same time of the day and on the Friday and the Sunday

into a nitrate or placebo group. Participants performed the same running training protocol during the study (described below). The participants completed a 60-s Wingate Test and a 10-km running TT at baseline and weekly during the training program. The Wingate test and the TT were conducted at the same time of the day and on the Friday and the Sunday of every week, respectively. This study has an experimental design similar to other studies already published by our group, including the study of Santana et al. [21].

Sports2019,7, 120 3 of 10 2.2. Participants Twenty healthy men were selected for this study. Participants with a minimum of 6 months of experience, with personal best times for 10 km between 55 and 65 min, and who trained 3 running sessions weekly were selected for the study. No supplements or ergogenics (di erent from the prescribed) were allowed during the study. Participants that performed less than 75% of the training sessions, that did not use supplementation as indicated by the authors, that changed their usual diet, and did not present a medical approval form in the rst week of the protocol were excluded. Four participants were excluded after the end of the study because they missed more than 25% of the training sessions (exclusion criteria). No participants dropped out of the study, but we analyzed only the 16 participants remaining (Table), performing a per-protocol set analysis. The University Ethical Committee approved the described experiments (protocol number CAAE: 38414814.3.0000.0089). All participants included in the study agreed to participate in the study and signed the consent form. Table 1.General characteristics of the participants at baseline. Characteristics Placebo ( n=8) Nitrate (n=8) p Age (years) 30.3 4.5 30.0 6.8 0.999 Height (m) 1.73 0.1 1.66 0.1 0.563 Weight (kg) 79.5 11.2 68.8 10.6 0.257 BMI (kg/m 2 ) 25.65 1.38 25.85 1.84 0.793 Fat free body mass (kg) 63.7 11.3 52.9 8.5 0.071 % Fat 20.4 4.5 23.3 2.4 0.176 2.3. Procedure 2.3.1. Experimental Protocol Nitrate and placebo supplements were supplied for 30 days using a double-blinded method. All supplements were produced in a compound pharmacy (Orion Compound Pharmacy-S ¢o Paulo-Brazil) that gave us bottles with A or B marked on it and then we randomized the distribution considering the performance times of the participants. The participants randomization was made by two members of our research lab (that were not part of this study) that o ered both types of bottles, initially to the 4 slowest runners (those closest to 65 min to complete the 10-kmTT) and so on, to the 4 fastest runners of the group. Once the participant chose a

of the participants. The participants randomization was made by two members of our research lab (that were not part of this study) that o ered both types of bottles, initially to the 4 slowest runners (those closest to 65 min to complete the 10-kmTT) and so on, to the 4 fastest runners of the group. Once the participant chose a bottle marked with a letter, he should only pick the same letter through the entire experimental protocol. We understand that this way, we had distributed equally and blinded both supplements through the participants' entire performance time interval and this distribution produced unbiased data. The participants that received the placebo (n=8) ingested 6 g of resistant starch in capsules, divided into three times a day, and the ones who received nitrate (n=8) ingested 750 mg of inorganic nitrate and 5 g of resistant starch in capsules, divided into three times a day. The participants received capsules with supplements (nitrate or placebo) each week during the intervention, totaling 30 doses in the month [21]. There was no di erence in the supplement consumption compliance. Both groups consumed more than 75% of the programmed supplementation in the month (nitrate 29.25 0.9; placebo 29.63 0.5 doses;p=0.402). 2.3.2. Wingate Test and Blood Lactate Concentration Participants performed a ve-minute pre-load with 80% of the maximum heart rate of each individual and, after that, they did a 60 s lead-in on a Wingate bike in order to generate the highest possible power in that period of time, with a load of 2% of the total body weight of the

Sports2019,7, 120 4 of 10 participants [22]. Participants were instructed to wear running clothes and the same running shoes in every test. [La ] was measured using a Roche portable lactate analyzer (Ho mann–La Roche, Basileia, Switzerland). The analyses were carried out immediately after the 60 s Wingate test. 2.3.3. Running Time Trial and Blood Lactate Concentration The 10-km running TT was performed two days after the Wingate test so there was no interference between the tests. Just as the Wingate test, participants were instructed to wear running clothes and the same running shoes in every test. Tests were executed at the same time, temperature, and climate conditions, according to the local weather forecast information, on an outside running track familiar to the participants. All tests started in the middle of August and nished in the end of September, taking around 45 days; this was the time of a reasonably steady climate condition. The tests were performed on a running track familiar to the participants, with all external factors interfering, to be as similar as possible to a running competition. A member of the study recorded time in the running test. Start and nish of the test were in the same place, with all the participants running together; they were not told to compete against each other but that happened, naturally. [La ] was measured as described, through the collection of a drop of blood from the ngertip on a reagent strip using a Roche portable Wingate test [21]. 2.3.4. Training Protocol Participants received a periodized training program of 4 weeks (30 days), involving three running sessions weekly (Monday, Wednesday, and Friday), totaling 12 training sessions in the month. On the rst training of each week, participants ran a moderate volume session (5 to 7 km). On the second day, the participants trained 4 to 6 sprints of 500 m at high speed with 2 min passive recovery between sprints. On the third day, the participants ran a long-distance (9 km to 12 km) session. Participants were supervised all day by the author of the study, and heart rate was

volume session (5 to 7 km). On the second day, the participants trained 4 to 6 sprints of 500 m at high speed with 2 min passive recovery between sprints. On the third day, the participants ran a long-distance (9 km to 12 km) session. Participants were supervised all day by the author of the study, and heart rate was monitored by the participants [21]. There was no di erence in the training sessions compliance. Both groups did more than 75% of the training sessions through the month (nitrate 11.75 0.5; placebo 11.63 0.7;p=0.598). 3. Statistical Analyses A 2 5 (group time) repeated measures analysis of variance with Bonferroni adjustment for multiple comparisons was used to compare placebo and nitrate group on the performance and [La]. Statistical signi cance was set atp<0.05. For all measured variables, the estimated sphericity was veri ed according to Mauchly's W test and the Greenhouse–Geisser correction used when necessary. The e ect size was also calculated via partial eta-squared for main e ect of time (ES). The data were analyzed using the Statistical Package for Social Sciences 17.0 (SPSSInc., Chicago, IL, USA). 4. Results Table mass, and % fat at baseline in the placebo and nitrate groups. There were no statistically signi cant di erences between groups at baseline for any variables investigated, reassuring the e ciency of the randomization protocol. Figure 60-s Wingate tests for placebo and nitrate groups.

Sports2019,7, 120 5 of 10Sports 2019, 6, x FOR PEER REVIEW 5 of 10 Figure 1. Comparison between placebo and nitrate groups for the time to complete the 10-km running time trials (panel 1-A), and maximum (panel 1-B) and average (panel 1-C) power developed during the Wingate test performed weekly during the training program. a is Bonferroni’s test result with p < 0.05 compared to baseline; b is Bonferroni’s test result with p < 0.05 compared to week 1. 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. Figure 1. Comparison between placebo and nitrate groups for the time to complete the 10-km running time trials (panel 1-A), and maximum (panel 1-B) and average (panel 1-C) power developed during the Wingate test performed weekly during the training program. a is Bonferroni's test result withp<0.05 compared to baseline; b is Bonferroni's test result withp<0.05 compared to week 1. We observed a main e ect of time (F=21.302,p<0.001, ES=0.68) and signi cant group time interaction (F=13.387,p<0.001) for the time to complete the TT. Post-hoc analysis revealed that time decreased signi cantly in all timepoints compared to baseline, and,

training program. a is Bonferroni's test result withp<0.05 compared to baseline; b is Bonferroni's test result withp<0.05 compared to week 1. We observed a main e ect of time (F=21.302,p<0.001, ES=0.68) and signi cant group time interaction (F=13.387,p<0.001) for the time to complete the TT. Post-hoc analysis revealed that time decreased signi cantly 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 di erence in the placebo group. There were no signi cant di erences 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 signi cant di erence between group were observed (F=3.028,p=0.104). There was, however, a signi cant group time interaction (F=2.934,p=0.028) for average power, but the post-hoc analysis did not identify signi cant di erence between groups. There were no signi cant di erences in the baseline values comparing both groups.

Sports2019,7, 120 6 of 10 Figure erences in the lactate concentration in the 10-km running test and the 60-s Wingate test weekly between placebo and nitrate groups.Sports 2019, 6, x FOR PEER REVIEW 6 of 10 Figure 2 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. Figure 2. [La − ] after the 10-km running time trial (TT) (panel 2-A) and the 60-s Wingate test (panel 2- B) for placebo and nitrate group. a is Bonferroni’s test result with p < 0.05 compared to baseline; b is Bonferroni’s test result with p < 0.05 compared to week-1; (*) Bonferroni’s test result with p < 0.05 compared to Placebo. 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. 5. Discussion 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 Figure 2. [La ] after the 10-km running time trial (TT) (panel 2-A) and the 60-s Wingate test (panel 2-B) for placebo and nitrate group. a is Bonferroni's test result withp<0.05 compared to baseline;b is Bonferroni's test result withp<0.05 compared to week-1; (*) Bonferroni's test

The study investigates the impact of nitrate supplementation on running performance in recreational runners.