Participants

The minimal required sample size was determined using G*power software [21]. The input parameters used for F test family were alpha = 0.05 and power = 0.90. Using the time until exhaustion during cycling at a supramaximal power output after control (157 ± 42 s) and the one preceded by drop jumps protocol (171 ± 44 s; de Poli et al. 2020), the minimal required sample size of 9 participants was estimated. A basketball team was contacted and fifteen young male basketball players (the players were from the under 17 (n = 7) and 19 (n = 8) categories and had experience of competing at a national level) were recruited to participate in the current study after meeting the following inclusion criteria: (1) basketball training experience ≥ 4 years; (2) absence of recent (< 3 months) musculoskeletal and joint injuries; and (3) absence of recent (< 3 months) regular use of any ergogenic substance (e.g., creatine, beta-alanine). The exclusion criteria were: (1) suffering an injury during the period of investigation (n = 2); (2) not tolerate the assessment procedures (n = 1); and (3) withdraw from the team (n = 1). Finally, 10 participants (age: 17.5 ± 1.2 years; stature: 1.91 ± 0.07 m; body mass: 87.2 ± 15.4 kg; competitive experience: 5.2 ± 1.5 years) were selected. Prior to the commencement of the study, the athletes were informed about benefits and risks of experimental procedures and signed an informed consent form. For the underage participants, their parents were informed and signed the consent form. All experimental procedures of the present study were approved by the Research Ethics Committee of the Sao Paulo State University (#2.540.512/2018) according to the Declaration of Helsinki.

Experimental design

A randomized cross-over design was adopted and all sessions were completed at the end of the pre-season and at the beginning of the competitive season, over a 3-week period. All assessments were performed on the same official basketball court, at the same time of day, with a consistent temperature of 29.3 ± 2.8 °C and a relative humidity of 52.2 ± 8.5%. All exercise sessions were separated with a minimum of 48 h and a maximum of 72 h.

Firstly, participants completed a familiarization session to the RSA test, to the CAs (i.e., drop jump and resisted sled towing), and to the neuromuscular assessments. In the following two sessions, participants initially performed a standardized warm-up (2 min of submaximal jogging, 1 min of side-to-side submaximal running, and 2 min of intermittent 10 m submaximal high-intensity run). After 5 min of passive rest, a protocol for the evaluation of neuromuscular function (i.e., measurement of central and peripheral fatigue, see below) was conducted. Following neuromuscular evaluation, participants performed one of the CAs (one trial for each CA) or remained at rest for 5 min (i.e., control condition). After interventions (4 min for drop jumps [considering the time spent during CA resulting in an interval ~ 5-min] and 8 min for heavy sled towing [considering the time spent during CA resulting in an interval ~ 8.2- min]), the RSA test was conducted with simultaneous recording of surface electromyography (EMG) of lower limb muscles. The reason for different intervals after CAs is reported in “Conditioning Activities” section below. Immediately after RSA testing, neuromuscular function was assessed. Finally, capillary blood samples were collected before each RSA test and at 3, 5, and 7 min post, to determine blood lactate concentration.

Repeated sprint ability test (RSA)

The RSA test, which included a change of direction (“L” format), used in this study consisted of 10 × 30 m maximal sprints with each sprint involving 5 rapid directional changes (6 runs of 5 m), interspersed with 30 s of passive recovery. This test was selected because it has been previously shown to mimic real game-play demands, with good reliability [2, 22]. All sprints were recorded by a 30 Hz digital camera (GoPro Hero 3 + Black, San Mateo, CA, USA) that recorded the start and the end of sprinting bouts. Subsequently, the recordings were analyzed using a custom-made software (v.0.8.15, Kinovea, Open source for Windows) to determine the sprinting time. The time to complete each sprint was used to determine the best time, mean time, slowest time, and total time (i.e., the accumulate performance time of 10 sprints only) of the RSA test.

Neuromuscular assessment

Force data acquisition

Participants performed two 5 s maximal voluntary contractions (MVCs) with the knee extensors of their self-reported dominant leg, interspersed with 1 min of passive rest [8, 23, 24]. The two MVCs were performed at baseline and as soon as possible after (77.50 ± 21.49 s) the RSA test. The measurements were carried out in a specific chair designed for maintaining hip and knee flexion at 90°, with participants secured by straps [8, 23, 24]. A metal rod was attached to the ankle and connected to a load cell with a maximum capacity of 100 kgf (MK Controle, São Paulo, SP, Brazil). The load cell signal was acquired by an analog data acquisition mode (NI 6009, National Instruments, Austin, TX, USA) sampling at 1000 Hz, using Labview software (National Instruments, Signal express, Austin, TX, USA). The recorded data were subsequently filtered by a second-order Butterworth filter and analyzed with custom designed software (MatLab R2015b, MathWorks, Natick, MA, USA). Before each test, the load cell was calibrated using known weights to create a linear regression model (r2 > 0.99). The peak force of MVCs was defined as the mean force recorded during 100 ms of the force plateau [8, 24].

Peripheral nerve stimulation

Peripheral nerve stimulation was delivered over the femoral triangle (cathode), and anterior to the greater trochanter of the femur (anode) with a high-voltage electric stimulator (Bioestimulador V2 400 V peak to peak, Insight, Brazil). A Ag/AgCl electrode (recording area, 78.5 mm2; Medi-trace, Dublin, Ireland) was used for cathode and a 5 × 5 cm electrode (Valutrode, CF5050 model, Brazil) used for anode [24]. The optimal intensity of stimulations was determined individually before each session by the application of consecutive incremental doublets to the relaxed muscle (square-wave, 100 Hz, pulse duration of 1 ms, initial intensity of 80 mA with 20 mA increments) until a plateau in force was reached [8, 24]. The maximal electrical current achieved (mA) was recorded, and supramaximal stimulation was ensured by increasing the final intensity by ~ 20% (253 ± 58 mA) [23, 24].

Doublet, high-frequency stimulation (i.e., square-wave, 100 Hz, pulse duration of 1 ms) were delivered during MVCs (~ 2 s), followed doublets at high frequency (Db100; stimulation frequency in Hertz) 5 s after MVCs, a single stimulus was delivered at 10 s and finally a low frequency stimulation (Db10; stimulation frequency in Hertz) was delivered at 15 s after MVC, with the muscle in a relaxed state [25]. The amplitude of the force signal superimposed by the doublet high frequency stimuli during MVCs, and the force produced by the high frequency stimuli at rest after MVCs were used to measure the percentage of voluntary activation (VA) through Eq. 1 [23, 24, 26]. The ratio between the force amplitudes produced by the doublets of low and high frequencies at rest, were subsequently calculated and used as an index of low-frequency muscle fiber depression [26].

$$% {text{VA}} = left{ 1 – [{text{Superimposed}};{text{force}} times left( {frac{{{text{Force}};{text{level}};{text{at}};{text{stimulation}}}}{{{text{Peak}};{text{force}}}}} right)/{text{high}};{text{frequency}};{text{force}}]right} times 100$$

(1)

Surface electromyography

A wireless EMG device (Cometa System, Italy) was used during the entire RSA test, while a fixed cable EMG device (Miotec, Brazil) was used during the neuromuscular assessments [24], with both devices sampling at 2000 Hz. The Ag/AgCl electrodes (recording area, 78.5 mm2; Medi-trace, Dublin, Ireland) were placed over the prepared skin (shaved skin and gently cleaned by abrasion with fine sandpaper and alcohol 70%) of the vastus lateralis (1/3 distal) to measure EMG responses during MVCs, with a ground electrode placed on the ulnar styloid process. During the RSA test, further electrodes were placed on the gastrocnemius medialis, rectus femoris, vastus lateralis, and biceps femoris as previously described [24].

The EMG signal was subsequently band-pass filtered (20–500 Hz). During the assessment of neuromuscular function, the root mean square (RMS) and median frequency (MdF) of 1 s of force plateau were used as indices of general neuromuscular discharge magnitude and rate, respectively [24]. The peak-to-peak maximal amplitude evoked by single stimulus (M-wave and [M-waveamp]) were calculated and used as indices of sarcolemmal excitability [24]. During the RSA test, all values of RMS and MdF were normalized by the first sprint value of the control condition. The RMS and median frequency were calculated using the LabChart Pro v.8 Software (ADInstruments, Colorado Springs, CO, USA).

Conditioning activities

Identification of optimal recovery interval for conditioning protocols

Prior to experimental sessions, pilot work with 6 physically-active males (age: 24.0 ± 2.5 years) was conducted to determine the optimal recovery interval (4 vs. 8 min) after both CAs, before completing the RSA test. For drop jumps, all the RSA test outcomes measured after 4 min of recovery were statistically better compared to control condition (RSA test performed without previous CA) (P = 0.048 for best time; P = 0.020 for mean time; P = 0.020 for total time, and P = 0.013 for slowest time) than those measured at 8 min of recovery (P = 0.508 for best time; P = 0.102 for mean time; P = 0.087 for total time, and P = 0.093 for slowest time) (see Additional file 1: Table S1), while for the heavy sled towing, only the mean time and total time were significantly lower compared to control condition after 8 min of recovery (P = 0.034 and P = 0.029, respectively) (see Additional file 1: Table S2). Therefore, the RSA test was performed 4-min after drop jumps, and 8 min after the heavy sled towing. All CAs were realized on the same court that RSA was performed.

Drop jumps

The drop-jump protocol consisted of 1 set of 5 repetitions, interspersed with 15 s of passive recovery [8, 14]. The drop-jump height was individually determined during the familiarization sessions. All participants performed 1 set with 3 jumps from four different heights (40, 60, 80, and 100 cm) on a force plate (Cefise, Nova Odessa, Brazil) and the box height selected was that exhibiting the highest reactive strength index (RSI; i.e., the ratio of jump height to ground contact time) [27].

Heavy sled towing

The heavy sled towing protocol composed of 1 sprint of 15 m with a sled attached to the waist, which was loaded with 75% of the players’ body mass [16].

Blood sample collection and analysis

Blood samples (25 µL) were collected at rest and 3 min after the RSA test from the earlobe, using heparinized capillary tubes. Blood samples were immediately deposited into microtubes containing 50 µL of sodium fluoride at 1%, and frozen at − 20 °C for posterior analyses (YSI 2900, YSI, Yellow Springs, Ohio, EUA).

Statistical analyses

The results are presented as means ± standard deviations and 95% confidence intervals (CI95%). A two-way repeated measures analysis of variance (ANOVA) was used to identify the effect of time and condition on neuromuscular assessment parameters. A repeated measures of ANOVA was used to compare RSA test performance outcomes and the percent changes of neuromuscular assessments between conditions. In all cases, the Mauchly´s test of sphericity was applied, and the Greenhouse–Geisser Epsilon correction was used when the sphericity criteria were not met. When necessary, the analyses were completed with SIDAK post hoc test. A significance level of P ≤ 0.05 was assumed in all cases. All statistical analyses were performed using the software SPSS version 20 (IBM Corp., Chicago, IL, USA). Cohen’s d effect size [ES(CI95%)] for the RSA outcomes and the delta changes between conditions for parameters of neuromuscular assessment was also calculated.

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