Participants undertook a 5-week resistance training program during which half the subjects took BA supplements according to whether they were assigned to a placebo group (PLA) or BA group. Before and after the training program, all participants performed a BS incremental load test at the laboratory under the same controlled environmental conditions. During the rest periods of this test, CMJ ability was monitored. The rest period from the pre-training BS test to the start of the training program was 72 h. Similarly, the rest period between the end of the training program and the post-BS test was also 72 h (Fig. 1).
Thirty young, healthy, resistance-trained men were enrolled in the study. All subjects were students of the Physical activity and Sport Sciences degree course at the Universidad Alfonso X El Sabio (Madrid, Spain). Exclusion criteria were: age younger than 18 or older than 25 years; 2) being an elite athlete; 3) having consumed any substance that could affect hormone levels or sport performance in the previous 3 months such as nutrition complements or steroids; 4) having consumed any narcotic and/or psychotropic agents, drugs or stimulants during the test or supplementation period; 5) any cardiovascular, metabolic, neurologic, pulmonary or orthopedic disorder that could limit performance in the different tests; 6) having less than 6 months of experience with BS training; 7) or less than 12 months of experience with resistance training; or 8) having a BS 1RM lower than 90 kg.
Subjects were randomly assigned to the two experimental groups: individuals in one group (n = 15) took BA and those in the control group (n = 15) were given placebo supplements (PLA) during the 5 weeks of training. Each day, it was ensured that each subject took the required supplement dose and attended the training sessions. At the end of the study, data were eliminated for subjects not completing all laboratory testing sessions, at least 85% of the training sessions, and/or missing three days or more of supplements. According to these criteria, the final study population was comprised of 26 subjects (age = 21.85 ± 1.6 years; weight = 80.27 ± 6.9 kg; height = 179.62 ± 6.1 cm; body mass index = 24.85 ± 1.8 kg·m2–1): 14 in BA and 12 in PLA.
At the study outset, participants were informed of the study protocol, schedule and nature of the exercises and tests to be performed before signing an informed consent form. The study protocol adhered to the tenets of the declaration of Helsinki and was approved by the Ethics Committee of the Universidad Alfonso X El Sabio.
Supplementation with β-alanine
The authors packaged and prepared the capsules containing the supplement or placebo. The capsules used were no. 1 opaque red (Guinama S.L.U, 0044634, La Pobla de Valbona, Spain) of capacity 0.50 mL/capsule corresponding to 400 mg of BA . For the encapsulation process, we followed the normalized working procedures, “Procedimientos Normalizados de Trabajo (PNT)” described for this purpose in the Formulario Nacional Español. The filling equipment used was a manual semiautomatic Carsunorm 2000 system (Miranda de Ebro, Spain).
Based on the doses used in other studies [35, 36], subjects assigned to the BA group were administered a daily β-alanine dose of 6.4 g taken as 8 capsules containing 800 mg at least 1.5 h apart and no longer than 3 h apart. The reason for the 8 daily capsules was to avoid the main side effect of paresthesia . Paresthesia is a mild sensation of prickling, numbness or burning in the skin that appears when doses of BA greater than 10 mg/kg are taken  and resolves 1 h after intake . Subjects in PLA took the same number of daily capsules containing sucrose. Only one of the authors was responsible for supplying the participants with the corresponding bottles of capsules. All subjects visited the research laboratory weekly to collect their supplement (BA or PLA) for that week. During each of the 5 weeks of the training program, the authors ensured each participant took their supplements and also guided each training session.
The 5-week training program was the same for the two groups BA and PLA. Three sessions were conducted per week (15 sessions in total) of around 35–60 min. Each day a register was taken and any participant missing more than 2 sessions (ie, around 15%) was excluded. In each session, after a 15 min warm up, three leg exercises were alternated as a circuit: back squat, barbell step ups and loaded jumping lunges (Table 1). Subjects performed a given number of repetitions of each exercise according to the allocated work time. In the first week, work time was 40 s per exercise and this was reduced by 5 s each week until a work time of 20 s (Table 2). Participants indicated their subjective exertion using the Borg scale of rating of perceived exertion (RPE) (CR-10) when completing each set of exercises and at the end of the session .
Load increases were guided by an observer during the program according to the perceived exertion of the previous week. In the first week, the load selected for the BS was 60% of 1RM obtained in the incremental load test before the start of the training program. In contrast, for the barbell step ups and loaded jumping lunges, the load was adjusted by each individual by targeting an RPE of 5–6 to complete 40 s of each exercise, thus maintaining around 50–60% of maximal intensity. Therefore, from the second week onwards: when RPE was 1 point below or above the target, the training load was increased or reduced respectively by 5% (kg) in each exercise; when between 1 and 2.5 points below or above the target, the load was adjusted by 10% (kg); and when 2.5 points above or below the target, the load was adjusted by 15%–20% [38, 39].
To increase the training volume, rest periods between exercises were reduced by 15 s per week from an initial 120 s to 60 s in the fifth week (Table 2). Rest periods between exercise sets were initially 2 min and then reduced by 15 s weekly until 1 min (Table 2). The numbers of exercise sets executed were 3 sets in week 1, 4 sets in weeks 2 and 3, and 5 sets in weeks 4 and 5.
Pre- and post-training test
For the pre- and post-training incremental load/CMJ test, subjects first undertook a general warm up followed by a specific warm up. The session commenced with 10 min of light to moderate trotting, 5 min of joint movement and ballistic stretching, and 1 set of 5 BS repetitions with a 20 kg load. During this set, subjects were instructed to increase execution velocity, targeting a velocity close to their maximum velocity in the final repetition. After 30 s of rest, subjects executed 3 consecutive CMJ at submaximal intensity. After 1 min of rest, subjects completed 1 set of 2 BS repetitions with 2 s of rest between repetitions, lifting a 30 kg load at maximum velocity of displacement for optimal muscle activation. After 30 s, subjects executed 2 CMJ at maximal intensity with 10 s of rest between jumps.
Back squat incremental load test
Three minutes after the warm-up, subjects started the incremental load BS test with an initial load of 30 kg. This load was increased in each set by 15 kg until average bar displacement velocity measured by a linear position transducer was under 0.7 m/s. Loads were then increased gradually in 1–5 kg steps until the 1RM was accurately determined. When mean velocities were above 0.7 m/s, subjects undertook 2 BS repetitions with a rest period between sets of 3 min. For lower velocities, only one repetition per set was executed with 5 min of rest.
The variables recorded in this session were average velocity (AV), peak velocity (PV), average power (AP), peak power (PP) and the load in kg lifted in the incremental BS 1RM test in which power output is at its maximum (Pmax) as follows :
Velocity (m·s− 1) = vertical movement of the bar (m) x time (s− 1).
Acceleration (m·s− 2) = vertical bar velocity (m·s− 1) x time (s− 1).
Force (N) = system mass (kg) × vertical acceleration of the bar (m·s− 2) + acceleration due to gravity (m·s− 2).
Power (W) = vertical force (N) × vertical bar velocity (m·s− 1). Power was calculated based on barbell velocity and not velocity of the centre of mass of the system [41, 42].
Back squat technique
For the BS, the subject stands with feet shoulder-width apart and the barbell placed on top of the shoulder blades with hands clutching the barbell, and then flexes the knees to 120° followed by their extension to the original standing position. Maximal strength, or 1RM, was defined as the maximum load the individual was able to lift with the appropriate exercise action .
The test was performed in a multipower, bar-guiding system Smith machine (Matrix, Chácara Alvorada, Brazil) using 20, 10, 5, 2.5 and 1.25 kg discs (Matrix). In this set up, both ends of the barbell are fixed allowing only vertical movement of the bar.
To estimate the execution velocity of each repetition in the incremental load test, we used a linear displacement system (Tendo Weight-lifting Analyzer System, Trencin, Slovak Republic). The cable was attached to one end of the bar to avoid hindering the BS movement. This system allows for measurement of the vertical displacement of the the bar according to the exercise movement and using the system’s software (Tendo Weightlifting analyzer 3.6.15), the device provides bar velocities (average and peak) and powers (average and peak) in the incremental load test .
Jump ability and muscular fatigue
At the start of the rest period for each set of the BS incremental load test, jump capacity was measured in 2 CMJs with 30 s of rest between one jump and the next. The variables jump height, power and take off velocity were measured using a Kistler Quattro Jump contact platform (Kistler Instruments, Winterthur, Switzerland). The CMJ test commences with the subject standing with the legs extended and arms on hips. The subject initiates the jump by bending the knees to ~ 900 (eccentric action) and immediately and synchronously then starts to extend the knees (concentric action) in an explosive movement to attain the maximum height possible. During the jump, the knees should be fully extended and contact with the ground is first made with the toes. Subjects were instructed to keep their hands on the hips during the jump and to avoid any sideways or backward/forward movements.
The effects of BA supplementation on the power output, kilograms lifted and movement velocity in response to the 5 weeks of training were assessed through a general linear model with repeated measures two-way analysis of variance as the Levene’s test revealed the homogeneity of variances of the initial variables and the Shapiro Wilk’s test confirmed their normal distribution. We thus considered an inter-subject factor (PLA, BA) and an intra-subject factor (pre-training, post-training) along with the effects of their interaction.
Although the general linear model with two-way analysis of variance revealed no significant differences between pre-training values for the two study groups, we performed a covariance analysis through a univariate procedure, in which the pre-training values were used as covariates to confirm that the differences observed in the general linear model were not due to differences in pre-training values betwee the PLA and BA groups.
To support the results of the previous analyses, we assessed the effect size of the kilograms lifted and number of sets accomplished. The effect size indicating the difference between means of the dependent variables was calculated using the formula: effect size = post – pre. For this analysis we also used a univariate general linear model.
In addition, the pre- and posttraining power and velocity data recorded at different work intensities in the BS incremental load test were compared through linear or polynomic regression models. We also determined through linear regression, the variables determining jump ability (jump height, average power and take off velocity) for different relative workloads in the BS incremental load test.
In all tests, effect size (ES) and statistical power (SP) were calculated. The general linear model procedure generates an effect size, known as partial eta squared, categorized as small = 0.01, medium =0.06, large = 0.14 . All data are provided as their means, standard deviation, and 95% confidence intervals (CI) when data are provided as percentages. Percentage improvements were calculated using the equation ([post - pre]/pre X 100). Significance was set at p < 0.05. All statistical tests were performed using the software package SSPS version 21.0 (SPSS, Chicago, Ill).