The experimental flowchart of the study is shown in Fig. 1. A randomized, three-trial, (placebo vs. WP vs. SP), cross-over, double-blind, repeated measures design was implemented. Initially, all participants underwent baseline testing, including assessment of their anthropometric profile (body mass, height), body composition, resting metabolic rate, daily dietary intake, habitual physical activity, physical conditioning level (maximal oxygen consumption, Yo-Yo intermittent endurance level 2, Yo-Yo intermittent recovery level 2) and technical skills (creative speed test and short dribbling test), over a 7-day period. Thereafter, they participated randomly (using an online semi-automated software) in three trials: (i) Placebo (PL), (ii) Whey Protein (WP) and (iii) Soy Protein (SP) supplementation.
Each trial included (i) an initial 1-week adaptive period (days 1–7), during which participants received individualized dietary plans providing them a daily protein intake of 0.8–1 g of protein/kg/day. This period included only very light-load soccer practice (consisted mainly of familiarization with the experimental training protocol) of limited duration (< 30 min per session) while participants were asked to abstain from any moderate-to-vigorous daily physical activity. (ii) a subsequent a 7-day pre-loading period (days 8–14), during which participants consumed the protein or placebo supplement (depending on the trial) on a daily basis. (iii) a 3-day experimental period, consisted of two speed-endurance training sessions performed 48 h apart (day 15 and day 17, respectively) with participants following the same dietary plan and supplementation protocol (protein supplement or placebo) as in the pre-loading phase. During the experimental period, participants were engaged only very light training of limited duration and avoided any moderate-to-vigorous physical activity as in the adaptive period. In-between the two speed-endurance training sessions (day 16), no training was performed. (iv) a 7-day washout period, during which participants were engaged only in daily very light training and followed the balanced dietary plan of the adaptive period. The washout period aimed at alleviating any inflammatory and muscle damaging effect induced by the previous trial prior to participation of the next one. The second training session was added to assess the effectiveness of the experimental treatments on the recovery kinetics of field performance.
Performance assessment (i.e. delayed-onset of muscle soreness, isokinetic strength, maximal voluntary isometric contraction, 10 m and 30 m speed, countermovement jump, repeated sprint ability) and resting blood sampling for the determination of biochemical indices related to exercise-induced muscle damage and redox status (i.e. creatine kinase, glutathione, total antioxidant capacity and protein carbonyls) were performed before each trial (pre; prior to the adaptive period) and at the end of pre-loading (post-load). By including the assessment at post-load we aimed at determining any potential alteration on dependent variables induced by WP vs SP supplementation per se. To assess the ability of WP and SP in enhancing recovery following speed-endurance training session 1, isokinetic strength, 10 m and 30 m speed, countermovement jump, repeated sprint ability were assessed at 24 h, maximal voluntary isometric contraction was tested at 1, 2, 3, 24 and 48 h (day 17, prior to speed-endurance training 2), while blood sampling and assessment of the delayed-onset of muscle soreness were performed at 24 and 48 hous (day 17, prior to speed-endurance training 2). Additional blood samples were drawn before and immediately after each speed-endurance training session for the determination of blood lactate concentration. During speed-endurance training sessions, field locomotor activity and heart rate responses were continuously monitored using global positioning system instrumentation and heart rate monitors.
Performance testing and blood sampling at all time-points were performed in the fed state and at the same time of day in each trial to prevent circadian rhythm variations. Similarly, speed-endurance training sessions were performed at the same time of day in each trial (17:00–18:00) under comparable environmental conditions (20–25 °C, ~ 60% humidity). Before each session, a standard breakfast and meal was consumed by all participants, and during each session they were allowed to consume only water ad libitum. Dietary intake was monitored daily during each trial.
A power analysis (effect size of 0.3, power of 0.80, probability error of 0.05, 2-tailed) for main variables and within-between factors repeated-measures analyses of variance, indicated a sample size of 8–10 participants. Accordingly, 12 soccer players were assessed for eligibility and 10 of them were finally included in the study (see Additional file 1). Participation was secured if volunteers (1) participated at a competitive level for ≥4 years (≥5 training sessions/week, ≥1 match/week), (2) were illness- and injury-free, (3) abstained (≥6 months before the study) from consumption of ergogenic supplements or medication and (4) were non-smokers. Participants’ characteristics at baseline are shown in Table 1. Experimental procedures were applied in alignment with the Declaration of Helsinki, as revised in 2013. Participants signed an informed consent after they had being informed about the goals of the study and its associated risks and benefits. The study was approved by the University of Thessaly Institutional Ethics Committee (1412/1–5/3–10/2018).
Speed-endurance training (~ 60 min) was performed on natural grass and incorporated 1 set of 8 (30-s each) maximum-intensity repetitions with a passive recovery of 2.5 min (work-to-rest ratio of 1:5) and utilized a soccer-specific drill, as described . The net exercise time of the protocol was ~ 4 min. Players were verbally encouraged during each repetition to perform at maximum intensity. In each session, speed-endurance training was preceded by a standard 15-min warm-up (shuttle running, dynamic stretching, agility drills) and followed by a 15-min cool-down (light-intensity running, passive stretching).
Dietary intervention and supplementation protocol
Participants’ daily energy and macronutrient intake were individually adjusted during the course of each trial based on dietary analysis, resting metabolic rate and physical activity-related energy expenditure (performed at baseline). Furthermore, given the reduced energy expenditure and training load of our participants throughout the study and the limited net exercise time of the two experimental exercise protocol, individual dietary plans were adapted to the current recommendations of the International Society of Sports Nutrition for individuals participating in fitness programs of low intensity and low volume  and as such total energy and macronutrient intake were much below the respective amounts recommend for soccer players during the in-season  (see Table 2). Accordingly, each participant received 28–29 kcals/kg/day (2190–2261 kcals per day), 3–3.6 g of carbohydrate/kg/day (43–52% of total energy) and 1.05–1.13 g of fat/kg/day (35–36% of total energy). Daily protein intake was set to 0.8–1 g of protein/kg during the adaptive period in an attempt to equalize the relative amount of protein received by participants prior to supplementation. To meet the study’s goal for increased protein consumption during pre-loading and the subsequent 3-day experimental period in WP and SP trials (in PL trial daily protein intake remained at 0.8–1 g/kg/day), protein intake was increased to 1.5 g/kg/day through supplementation, as recommended for individuals engaged in moderate amounts of high-intensity training .
Supplementation during the three trials (i.e. throughout pre-loading and the 3-day experimental period) included WP isolate (Instantized BiPRO® I.P., Davisco Foods International, INC, Minnesota, USA – WP: 91 g; carbohydrates: 0 g; fat: 1.3 g; 380 kcal per 100 g), SP isolate (Soy Protein Isolate, My Protein, UK – SP: 93 g; carbohydrates: 1 g; fat: 3 g; 379 kcal per 100 g) or isoenergetic placebo (Maltodextrin, My Protein, UK) in a random order. Specifically, during pre-loading and the day in-between training sessions (day 16), drinks were consumed in a single dose with breakfast, whereas on training days (days 15 and 17), drinks were administered immediately post-training as a single bolus. The protein content in each protein drink was individually adjusted, based on each participant’s dietary protein intake, to account for a total daily protein consumption of 1.5 g/kg/day. All drinks were isovolumetric (~ 500 ml), consumed with water and flavored with ~ 10 drops of non-caloric sweetener (Flavdrops chocolate, My Protein, UK) to make the contents indistinguishable and non-transparent. Participants were asked 9 times each (once/day) if they knew what was the ingested solution. Out of a total of 270 responses, 129 times answered “I do not know”, 85 times answered incorrectly and 56 times answered correctly (possibly due to chance). Hence, we believe that participants were well-blinded. During trials, participants adhered to a daily nutrition plan that included three meals and two snacks. Daily protein intake was derived from dairy, eggs, poultry, meat and nuts.
Diet monitoring and analysis
A registered dietitian instructed participants how to record food/fluid servings and their daily dietary intake. Then, they completed 7-day diet recalls to evaluate their macronutrient and energy intake using a nutritional software (Science Fit Diet 200A, Science Technologies, Athens, Greece) as previously described [16, 17]. Participants’ daily dietary intake was also monitored and analyzed during all trials and the wash-out periods to ensure that there were no deviations from the prescribed diet.
Field activity and internal load during speed-endurance training was monitored using high time resolution global positioning system equipped with heart rate monitors (10-Hz GPS, 200-Hz triaxial accelerometry; Polar Team Pro, Polar Electro, Kempele, Finland) as previously described . Field activity was classified as total distance; average and maximum speed; high-intensity running (distance covered at speeds 14–21 km/h); high-speed running (distance covered at speeds > 21 km/h); intense accelerations and decelerations counts (> 2 m/s2). Internal load was expressed as maximum and average heart rate.
Body mass and height were measured on a beam balance with a stadiometer (Beam Balance Stadiometer, SECA, Vogel & Halke, Hamburg, Germany) as described . Dual-energy X-ray absorptiometry (DXA, GE Healthcare, Lunar DPX-NT) was utilized for body composition assessment as previously published . Open-circuit spirometry with an automated online pulmonary gas exchange system (Vmax Encore 29, BEBJO296, Yorba Linda, CA, USA) was used for the assessment of maximal oxygen consumption, via breath-by-breath analysis during a graded exercise testing on a treadmill (Stex 8025 T, Korea) as previously described . For resting metabolic rate measurement, resting VO2/CO2 values were measured in the morning (07.00–09.00) after an overnight fast using an open-circuit indirect calorimeter with a ventilated hood system (Vmax Encore 29, BEBJO296, Yorba Linda, CA, USA) and the 24 h-resting metabolic rate was calculated as previously described . Physical activity levels were monitored over a 7-day period via 3-axial accelerometers (ActiGraph GT3X+, Pensacola, FL, USA) as described . Soccer-specific conditioning was measured using the Yo-Yo intermittent endurance level 2 and Yo-Yo intermittent recovery level 2 tests as previously described . Participants’ level of technical ability was determined using the creative speed and short dribbling tests as described elsewhere .
Maximal voluntary isometric contraction as well as concentric and eccentric isokinetic peak torque of the knee extensors and knee flexors (at 60°/s) were measured on an isokinetic dynamometer (Cybex Norm 770; Cybex, Ronkonkoma, NY), both in dominant and non-dominant limb, as described . Countermovement jump was measured on an Ergojump contact platform (NewTest Ltd., Kiviharjuntie, Finland) as described . Sprint time over 10- and 30-m, and repeated sprint ability fatigue index assessments were performed using photocells . Delayed-onset of muscle soreness of the knee extensors and knee flexors of the dominat limb was assessed by palpation .
Blood sampling and assays
Fasting blood samples were collected by venipuncture using a disposable 20-gauge needle from an antecubital arm vein with the participants seated. For serum separation (to measure creatine kinase and total antioxidant capacity), samples were collected in tubes containing SST-Gel/clot activator and allowed to clot at room temperature for 30 min before centrifuged (1370 g, 4 °C, 10 min). For plasma separation (to measure protein carbonyls), samples were collected in tubes containing ethylenediaminetetraacetic acid and centrifuged immediately (1370 g, 4 °C, 10 min). Red blood cell lysates were prepared (to measure hemoglobin and glutathione) after lysis of packed erythrocytes following plasma separation. Samples were stored at − 80 °C in multiple aliquots until assayed. Samples were protected from light and auto-oxidation and thawed once before measured in duplicate.
Blood lactate concentration was assessed using a hand portable analyzer (Lactate Plus; Nova Biomedical, Waltham, MA) as described . Creatine kinase was measured using an automated Clinical Chemistry Analyzer Z1145 (P. Zafiropoulos S.A., Athens, Greece) with commercially available kits (P. Zafiropoulos S.A.) as previously described . Glutathione, total antioxidant capacity and protein carbonyls were analyzed spectrophotometrically as previously described . Spectrophotometric assays were performed on a Hitachi 2001 UV/VIS (Hitachi Instruments Inc., U.S.). The inter- and intra-assay coefficients ranged from 1.5 to 7.2% for all assays.
Data are presented as means±SD. Normality was examined using the Shapiro-Wilk test. Performance, muscle damage and redox status variables were analyzed using a two-way (condition vs time), repeated-measures ANOVA with planned contrasts on different time points. When a significant interaction was detected, a Bonferonni test was applied for post-hoc analysis. Percentage change from speed-endurance training 1 to 2 (Δchange) of field activity data in each trial were analyzed using a one-way ANOVA with a Bonferroni correction for multiple comparisons. For all dependent variables, effect sizes and confidence intervals were calculated according to the corrected for bias Hedge’s method (see Additional file 2). Effect sizes were considered as none, small, medium-sized and large for values 0.00–0.19, 0.20–0.49, 0.50–0.79 and ≥ 0.8, respectively. The IBM SPSS Statistics for Windows was used for analyses (version 20; IBM Corp., Armonk, NY) with significance accepted at P ≤ 0.05.