Experimental design
Firstly, the participants completed two weeks of familiarization with the exercise program routine. On the first visit to the laboratory, the participants were assessed for anthropometrics and body composition. On the second visit, 24 h later, the one repetition maximum test (1RM) was performed. Next, participants were pair-matched based on initial fat-free mass, fat mass and strength levels, and then randomly allocated to one of two treatment groups (CAP or Placebo). Food records were distributed to all participants pre and post-intervention to record food intake for three nonconsecutive days (one weekend and two weekdays), where one weekday corresponded to the day before the acute resistance exercise session. The third visit was separated by 72 h and subjects performed the acute resistance exercise session test. Blood samples were collected 90 min post-prandial (rest) and immediately following the acute resistance exercise session test. After 48 h, both groups participated in a 6-week progressive resistance training program, as shown in Supplementary Table 1, combined with placebo or CAP supplementation, and then returned to laboratory at post-training to repeat all assessments (4th to 6th visits). Supplementation continued through the post-training evaluation phase. The experimental design is illustrated in Fig. 1.
Participants
This randomized, placebo-controlled and double-blind study was carried out according to the 2013 Revision of the Declaration of Helsinki and it was approved by the Institutional Review Board from Federal University of Piaui in February, 2019 (Protocol number: 3.169.545) according the ethical standards [12]. Participants were invited through media and newspaper advertising at campus to participate in the study. The participants contacted the researchers by phone and an appointment was made in order to carry out a more detailed interview.
The inclusion criteria for the present study were: (1) males between 18 and 30 years of age; (2) had not participated in regimented resistance training in the previous 6 months; (3) had not used any ergogenic supplement for at least two years prior to the study; (4) did not smoke or drink alcohol within of testing visits (1st to 3rd visit at baseline and 4th to 6th visit at post-training); (5) no contraindications involving the cardiovascular system, muscles, joints, or bones of the lower and upper limbs that could limit exercise. All participants signed a consent form and were informed about the purpose of the study and the possible risks. During the study, all participants were instructed not to use any other supplement or ergogenic substance and to maintain their current diets. Additional exclusions criteria included missing more than 3 workouts during the intervention.
Out of a total of 58 men who participated in the first screening, 28 met all the inclusion criteria and 22 agreed to participate in the study protocol. This study used a randomized, double-blind design. Participants were pair-matched based on initial fat-free mass, fat mass and strength levels and then randomly allocated to one of two treatment groups: CAP (n = 11) and placebo (n = 11). During the intervention, one participant from placebo group dropped out of the study due to unspecified reasons and there was an outlier in the same group who was excluded from the final analysis due higher fat mass in relation to sample. It was calculated as the ratio Z as the difference between the outlier and the mean divided by the SD (via (https://www.graphpad.com/quickcalcs/grubbs1/) and the outlier comes from a different population if Z is greater than 1.96. Thus, the final sample analyzed was 11 subjects in the CAP group and 9 in the placebo group.
Procedures
Dietary intake assessment and supplementation procedure
Food records were distributed to all participants pre and post-intervention to record food intake for three nonconsecutive days (one weekend and two weekdays), where one weekday corresponded to the day before the acute resistance exercise session. A breakfast (total energy 603 ± 65 kcal, consisting of 25 % protein, 50 % carbohydrates and 25 % fat) was provided to participants before the acute resistance exercise (1 h 30 min). The software Dietbox (version 3.3.0) utilized the database of Brazilian food composition table (TACO) to calculate dietary intake. The volunteers were instructed not to consume chili peppers or other spicy foods and any kind of supplementation during the study, as well as coffee, tea, alcohol and/or stimulant drinks for a period of 12 h prior to assessments.
Each participant randomly consumed 2 capsules of placebo (starch) or CAP with 6 mg Capsiate per capsule (12 mg Capsiate in total), which were identical in appearance to ensure a double-blind design, 45 min before each experimental test and before lunch on non-training days. This time was selected because capsaicin reaches maximum concentrations within 45 min after ingestion [12]. CAP was standardized to contain 50 % Capsiate (Capsicum annuum L.) (Purifarma-Gemini Pharmaceutical Industry Ltda, Anapolis, GO, Brazil). Study products were delivered to each individual subject by a person who was not directly involved in the data collection to ensure blinding.
Anthropometric and body composition measurements
Body weight was measured using an electronic scale (Filizola PL 50, Filizzola Ltda., Brazil), with a precision of 0.1 kg. Height was measured on a fixed stadiometer with an accuracy of 0.1 cm and a length of 2.20 m. The total body water (TBW), intracellular (ICW) and extracellular water (ECW), fat-free mas (FFM), fat mass (FM), and percentage of fat mass (FM%) were assessed using a spectral bioelectrical impedance analysis and accompanying software (InBody S10, Gangnam-gu, Seoul, Korea). Based on results of a small pilot study (n = 8), the test retest intraclass correlation coefficient (ICC) from our lab were TBW (0.99), ECW (0.99), ICW (0.98), FM (0.97) and FFM (0.99).
Blood samples and analyses
Blood samples (20 ml) were collected at pre-training and after 6 weeks of training at rest and immediately post-exercise (within 5 min) during the acute resistance exercise session. The tubes were centrifuged at 3000 rpm for 15 min at 4ºC, and plasma and serum samples were stored at -80ºC until analysis. The Tumor Necrosis Factor-Alpha (TNF-α), soluble TNF-α receptor (sTNF-r), IL-6 and IL-10 serum concentrations were determined by enzyme-linked immunosorbent assay (ELISA) technique using high detection sensitivity kits (R&D System®, Minneapolis, MN, United States) with range between 15.6 and 1000 pg/mL for TNF-α, 7.8–250 pg/mL for sTNF-r, 3.13–300 pg/mL for IL-6 and 7.8–500 pg/mL for IL-10; and intra- and inter-assay variation (%) of 4.2–5.2 and 4.6–7.4 (TNF-α), 3.6-5.0 and 3.7–8.8 (sTNF-r), 1.6–4.2 and 3.3–6.4 (IL-6) and 1.7-5.0 and 5.9–7.5 (IL-10), respectively.
Muscular performance tests and resistance training protocol
Prior to all pre-training muscular performance tests, the participants completed two weeks of low-intensity familiarization with the exercises listed in Supplementary Table 1 with 2 sets of 15 repetition in each exercise and 60 s of rest. Subjects completed a warm-up before each test, which consisted of 5 min of walking (~ 6 km.h− 1) and a subsequent set of 10 repetitions at approximately 50 % of the 1RM for the first exercise of the specific workout of the day, according to Supplementary Table 1.
Lower and upper body strength was assessed by 1RM testing in the 45º leg press exercise (1RM-leg press) and the bench press (1RM-bench press) exercises, according the National Strength and Conditioning Association [13].
The acute resistance exercise session consisted of 3 sets of 45º leg press followed by 3 sets of bench press until momentary muscular failure with a load corresponding to 70 % of the 1RM and 90 s of rest between sets and exercise, according to Conrado de Freitas et al. [6] Both peak and mean power output were recorded for each repetition using the Tendo™ Power Output Unit (Tendo Sports Machines, Trencin, Slovak Republic) and the peak power for 3 sets (watts) and mean power for 3 sets (watts) were used [14].
The chronic resistance training program consisted of two progressive phases (Supplementary Table 1) and the intensity was controlled by zone of repetition. The sets were executed until concentric muscular failure (i.e. when the participants performed the training with repetitions varying from 10 to 12 RM, they were to execute at least 10 and no more than 12 RM). In the case of the participants executing more repetitions, the load was increased in order to keep within the training zone [15]. Fitness professionals supervised all testing, the exercise routine was conducted face to face (one fitness professional trained two participants), and the total number of repetitions performed was recorded for each set and each exercise, and the total weight lifted was calculated (repetitions × weight lifted × sets). The sum of the total weight lifted for each day was calculated to show total volume per week across the study.
Statistical analysis
Using a partial eta-square (0.26) for fat-free mass in the present study, and an alpha value of 5 % via GPower 3.1 software, a 99 % power to our sample size was found by analyzing the difference between groups post hoc. For the outcome measures, an Analysis of covariance (ANCOVA) was applied to adjust for pre-intervention and an Independent t test was used to verify the difference between relative changes (∆%= post-exercise value minus rest value/rest*100) and confidence interval (CI 95 %) was calculated. For the acute resistance exercise analysis, a mixed between–within subject repeated measures ANOVA was used where the supplementation group (placebo vs. capsaicin) was included as the between-subject factor, exercise effect (rest vs. post-exercise) and time (pre-training vs. after 6-weeks of training) were used as the within-subject factors. The inflammatory data was transformed to log, since the data presented non-parametric distribution, according to the Shapiro-Wilk test and a mixed between–within subject ANOVA was used again. The estimated sphericity was verified according to Mauchly’s W test and the Greenhouse-Geisser correction was used when necessary. Effect sizes for the ANOVA were calculated using partial eta squared (η2) for time, group and interaction. Data are presented as mean and standard deviation (SD). Statistical significance was set at p < 0.05. The data were analyzed using the Statistical Package for Social Sciences 17.0 (SPSS Inc. Chicago. IL.USA). In addition, it is important highlight that statistician was blind to both groups during data analyses.