Experimental design
This study was a randomized, crossover, double-blind and placebo-controlled study with experimental trials conducted in the morning (6 to 9 AM) under similar weather conditions (relative humidity: 60-90%; wind: 5-29 km.h-1; temperature: 19-26°C; altitude: 475 meters) to minimize the chronobiological variance. The participants were instructed about the protocol and were requested to maintain their training and nutritional habits during the study. The participants visited the track for two 10-km running tests under CAP or placebo conditions separated by one week. The trials were performed on a 400 meter official outdoor track, between March to May (in total, it took eight weeks to have all data collected completed).
Participants
They were advised to abstain from chili peppers or other spicy foods, caffeinated, supplements or ergogenic substances, energy drinks (which contain any kind of stimulant ingredient) or alcoholic beverages as well as strenuous physical exercise within 24 hours prior to testing. Subjects were requested to maintain their regular food intake, and to maintain the same physical exercise regimen 48 hours prior to testing. Additionally, they were instructed to consume breakfast at home as usual, before each experimental trial. Overall energy consumption (kilocalories) and macronutrient intake was calculated from the Brazilian food composition table (TACO) to ensure a similar intake in both experimental trials. The Research Ethics Committee (protocol number 3.654.560/2019) approved the study protocol which was conducted according to the 2013 Revision of the Declaration of Helsinki. The subjects signed a consent form and were informed about the purpose of the study and possible risks.
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Procedures
Supplementation protocol
A researcher who did not participate in the study (double-blinded) randomized the participants by a random sequence generator (www.Random.org) in placebo and CAP condition trial. The second trial was performed in a cross-over manner. In both conditions, subjects ingested two capsules of 12 mg of CAP (24 mg) or placebo according to our previous study that demonstrated benefits of such an application rate [26]. Participants ingested one capsule 45 minutes before the test and the second one immediately before the start of the 10-km running test. This strategy of CAP supplementation was applied to use peak concentration occurring at about 45 minutes after intake and a bioavailability of approximately 25 minutes, to remain the plasma level elevated up to 105 minutes [17, 18]. The product used for this study contained 50% extract from capsicum (capsicum annuum L.) from India (Purifarma-Gemini Pharmaceutical Industry Ltda, Anapolis, GO, Brazil) and 50% of maltodextrin. The correction factor in assay calculation was used by Pharma Nostra (Campinas, Brazil) to guarantee 100% of capsinoids (Capsiate) in each capsules of 12 mg. The capsules were identical and without flavour. It was delivered to each subject by an independent person who did not participate in the research team in order to secure double-blinding.
10-km time-trial running test
All participants were experienced in long-distance running and familiar with the track where the tests were performed. All subjects performed the trials 1 and 2 without the presence of opponents or another competitor on the track. Each trial was preceded by a similar self-determined warm-up of 10 min. All participants were encouraged to give their best performance and were cheered by the research team continuously. Participants freely choose their pacing strategy during the run and the split times were recorded every 400 meters. The overall mean velocity for each trial was calculated by dividing the total distance covered by the total time of the test. All subjects had access to mineral water during the run ad libitum.
Blood lactate
Twenty-five microliters of blood were collected from the volunteer’s right ear lobe before, immediately after, and 3, 5 and 7 minutes after each trial. The lactate concentration was determined by electroenzymatic methods using an automated analyser (YSI 2300 STAT®, Yellow Springs, Ohio, EUA). Peak lactate concentration was defined for each participant as the highest post-exercise lactate concentration value [27].
Rating of perceived exertion and heart rate
Rating of perceived exertion was evaluated by the 6-20 points Borg scale and peak of heart rate (Polar® Vantage NV, Electro Oy, Finland) was recorded immediately after the 10-km run was completed.
Incremental test
To determine aerobic fitness, the participants performed a maximal incremental test on the treadmill (Inbramed ATL®, Porto Alegre - Brazil) until exhaustion. Maximum oxygen uptake (V̇O2max) was determined as the mean of the latest 30 seconds from the last stage completed in the incremental test. Gas exchange variables were measured breath-by-breath using a gas analyzer (Model Quark PFT Ergo – Cosmed® – Rome, Italy). Before each test, the gas analyser was calibrated according to the manufacturer’s recommendations. The participants performed three minutes of warm-up at 8 km.h-1. Each stage of the test lasted one minute and the first stage was performed at 9 km·h-1, with speed increments of 1 km·h-1 per stage until voluntary exhaustion [28]. The maximal velocity (Vmax) was assumed as the highest velocity in the stage completed before exhaustion in the incremental exercise test. Mean Vmax was compared to the mean velocity of the 10-km running time trial test (Table 2).
Body composition and anthropometry
All the procedures were performed by the same person in an acclimatized room. Body mass was measured on an electronic scale (Filizzola® PL 150, Filizzola Ltda, Brazil). Body height was measured with a wall-mounted stadiometer (Sanny®, São Paulo, Brazil). Fat-free mass and fat mass were estimated by bioelectrical impedance following the procedure of the manufacturer (Bia Analyzer TM®, The Nutritional Solutions Corporation, Harrisville, MI, USA).
Statistical analysis
The sample size of this study was calculated according to previous studies from our research group that verified the improvement of running time in CAP conditions [13, 14]. An effect size of 0.60 as well as β-1 = 0.85 were considered for the power analysis using G*Power software. A sample size of n = 21 was calculated to sufficient to detect the prescribed effects within this study protocol.
Data were analysed using Statistical Package for the Social Sciences (SPSS® v.24, Inc., Chicago, IL, USA). Data normality was verified using the Shapiro-Wilk test. Data are reported as means and ± standard deviation (SD). The intraclass correlation coefficient (ICC) test was applied to verify the reliability between trial 1 and 2 (blinded). A paired t test was used to compare both conditions (CAP and placebo) in case of normally distributed data. Statistical significance was set at P < 0.05.