Dietary protein optimizes resistance training adaptations for muscle mass accretion [1,2,3]. Greater protein requirements for athletes and active individuals training to build muscle, combined with complex schedules and/or lifestyles may compromise nutrition strategies that enhance muscle hypertrophy over time. Total daily intake of protein appears to be the most potent factor in maximizing muscle adaptation in conjunction with resistance training [4]. However, the concept of protein timing implies the peri-workout and pre-sleep time periods may have a special role in optimizing dietary proteins for athletic purposes [5, 6].

The potential role of nighttime nutrition in muscle adaptation to training is often overlooked. However, during sleep, ingested protein is digested and absorbed equivalent to non-sleeping periods [7, 8]. Muscle and other tissues respond to hyperaminoacidemia during sleep by increasing muscle protein synthesis (MPS) when prior resistance exercise occurs in the evening [7, 8]. In a 12-week study, the combination of evening resistance training and supplemental casein protein lead to greater gains in strength and muscle mass than resistance training alone [6]. While the resistance training stimulus was consistent across all participants, the group receiving the supplemental casein had a significantly greater daily protein intake (1.9 vs. 1.3 g • kg− 1 • d− 1), as the control group did not receive an isocaloric or isonitrogenous comparator. Furthermore, since the training stimulus was during the evening, followed by supplemental protein, influential elements of protein timing might exist. Thus, one of the purposes of this study was to determine whether increased protein intake would be equally effective if the protein was consumed before bed, versus earlier in the day and closer to an earlier training stimulus.

Athletes are often hesitant to eat late into the evening due to the perception that it would disrupt body fat breakdown during sleep and, in turn, leanness. However, single night studies involving supplemental protein prior to bed suggest that this action may not significantly disturb lipolysis and fat oxidation overnight [9,10,11]. Since protein consumption closer to sleep likely does not influence body leanness, such a practice can help with daily planning and achieving appropriate levels of dietary protein. Limited research has been performed examining exercise training and supplemental protein on potential changes in muscle size and performance and adipose tissue simultaneously. In one study, males and females already engaged in unsupervised exercise training were provided 54 g of casein protein, either at night or in the morning, for 8 weeks [12]. Protein intake was increased in both groups from 1.7–1.8 to 2.4 g • kg− 1 • d− 1 and no differences in strength and body composition from beginning to end were observed. Yet to date, an incremental, high-intensity, monitored training program has not been conducted in conjunction with supplemental casein protein (night vs day) on measures of muscle thickness, body composition and strength. Thus to our knowledge, this is the first longitudinal isonitrogenous, isocaloric, nighttime casein supplementation study investigating the impact on body weight (BW) and composition as well as strength and muscle hypertrophy when an impactful resistance training stimulus occurs earlier in the day. It was hypothesized that the nighttime (NT) supplemented group would experience greater benefit to resistance-training induced physiological changes. The results of this study are important to athletes and active individuals who train for performance, aesthetics, and health.

Methods

Experimental design

In a randomized, double-blind, placebo-, diet-, and exercise-controlled trial, participants in the NT group were supplemented with 35 g casein protein at night immediately before going to sleep and 35 g maltodextrin earlier in the day, and participants in the daytime group (DT) were supplemented with 35 g maltodextrin at night immediately before going to sleep and 35 g casein protein earlier in the day. Participants were randomized to the NT or DT group by stratified randomization based on cross-sectional area of the rectus femoris (CSA) to balance groups based on muscle size and strength. The supplement taken early in the day was not consumed within 3 h of beginning or ending exercise, nor was it consumed within 6 h of sleep. Exercise programs and diets were prescribed for each participant, and both were supervised and tracked throughout the intervention. Prior to and following the intervention period, participants’ body composition, muscle hypertrophy, and athletic performance were assessed.

Participants

Healthy, recreationally active, 18–25-year-old males (NT: 71.4 ± 11.1 kg, 170.2 ± 3.8 cm training 4.0 ± 0.9 days/week for prior 2.7 ± 0.52 years; DT: 79.5 ± 21.5 kg, 178.1 ± 6.5 cm training 3.7 ± 1.1 days/week for prior 2.0 ± 0.82 years) were screened for participation. Individuals were eligible if they engaged in regular exercise for the previous 1–3 years at a frequency of 2–5 days per week and were excluded for tobacco use, excessive alcohol intake (≥ 12 drinks/week), having history of medical or metabolic complications, as well as use of nutritional supplements or medications that would significantly affect study outcomes. Study protocols were approved by the Institutional Review Board at Texas Woman’s University, and informed consent was provided by all participants prior to the investigation.

Measurements

BW was assessed using a physician’s scale (BWB-800, Tanita Corporation, Tokyo, Japan) and height by stadiometer. Dual-Energy X-ray Absorptiometry (DXA; Lunar Prodigy, General Electric Corporation, Boston, MA) was used to assess fat mass (FM), body fat percentage (BF%), lean soft tissue (LST), and appendicular LST (ALST). Test–retest reliability for DEXA measurements in15 subjects, resulted in an average intraclass correlation (ICC) of > 0.99. Ultrasonography-determined (Logiq e, General Electric Corporation, Boston, MA) CSA of the rectus femoris and combined muscle thickness (MT) of the vastus lateralis and vastus intermedius were measured as previously described [13]. Briefly, CSA measurements were conducted on the anterior thigh at 75% femur length, defined as the distance from the anterior superior iliac spine to the superior aspect of the patella, and MT measurements were conducted at 50% femur length, defined as the distance from the greater trochanter to the lateral epicondyle of the femur. Test–retest reliability for ultrasound measurements, as determined using 5 subjects, resulted in an average ICC > 0.99. All body composition measurements were conducted in the morning following an overnight fast while the participant wore only lightweight athletic shorts, a t-shirt, and socks. Leg press and bench press 1-repetition-maximum (1RM) testing determined changes in strength. Participants were required to allow the sled to descend to a knee angle of 900 and press back to the starting position for a successful attempt in the leg press 1RM. In the bench press 1RM, they were required to touch the bar to their chest without bouncing and press back to the starting position without lifting their hips from the bench. A standard 3-min or 5-min rest period was used between all warm up sets or 1RM attempts, respectively. Participants first warmed up with the bar, followed by an initial weight equal to approximately 50% of 1RM and increased intensity progressively over 3–4 sets up to ~ 85% 1RM for a single repetition prior to beginning 1RM attempts at ~ 90% estimated 1RM. During mid and post 1RM testing, the final warm up set was performed at an intensity 2.3 kg less than their previously determined 1RM for one repetition, and participants first 1RM attempt was performed at an intensity 2.3 kg more than their previously determined 1RM. Thereafter, intensity was increased by 2.3–22.7 kg per attempt according to participants’ apparent capabilities observed by qualified researchers. Vertical jump (VJ) testing was conducted to assess changes in jump height (Vertec, Perform Better, Cranston, RI), during which peak power (PP) and velocity (PV), average power (AP) and velocity (AV), and peak force (PF) were determined using a linear force transducer (Weightlifting Analyzer, Tendo Sports Machines, Slovak Republic) fastened to the back of a thin canvas belt tightened over top participants’ waistbands during their jump attempt. Participants were weighed fully clothed for accurate loads to be entered into the force transducer. Test-retest reliability for performance measures, as determined using 4 participants, resulted in an average ICC > 0.96. All pre and post testing measures were conducted at the same time of day to prevent diurnal variations. Delayed onset muscle soreness (DOMS) and rating of perceived exertion (RPE) were measured at the beginning and end of each exercise session, respectively, using a 10 cm visual analogue scale.

Resistance training protocol

Exercise sessions took place at training facilities on campus and were monitored by National Strength & Conditioning Association Certified Strength & Conditioning Specialists. The exercise stimulus was a periodized resistance training intervention consisting of two 5-week mesocycles, which trained each major muscle group twice weekly. Within each mesocycle, intensity increased as repetitions decreased (Table 1). During week 5, the upper and lower body strength oriented training sessions began with 1RM testing to more accurately prescribe training loads during the following mesocycle. During week 10, the Thursday and Friday sessions were composed of only a warm-up to 1 repetition with each participant’s 1RM from week 5. Intensity and number of sets and repetitions were recorded during every training session.

Table 1 Resistance Training Schedule Full size table

Diet and protein supplementation

Caloric targets were established by Mifflin St. Jeor equation with a 1.6× adjustment for activity with 1.8 g protein/kg BW inclusive of the casein supplement, and the remainder of calories were provided as 35% fat and 65% carbohydrate. Total daily energy intake ratios corresponded to 20% protein, 52% carbohydrate, and 28% fat. Dietary compliance was monitored throughout the study by weekly intake surveys recorded using commercially-available software (MyFitnessPal, Baltimore, Maryland). Participants met weekly with researchers to assist them in reaching their dietary goals. Twenty-five grams of whey protein (ISO100, Dymatize, Dallas, TX) was provided to all participants post workout. Casein (as calcium caseinate; Friesland Campina, Amersfoort, The Netherlands) and maltodextrin supplements were flavor- and color-matched by the research staff. Each serving of casein provided 35 g of protein, < 0.5 g of fat, and < 0.5 g carbohydrate (lactose), and each serving of maltodextrin provided 35 g of carbohydrate, < 0.5 g fat, and < 0.5 g protein. Participants were provided canisters of casein and placebo at weeks 0, 3, and 6 with a supplement log to record time of consumption each day. Canisters were weighed before and after being given to the participants as a confirmatory measure of compliance.

Statistical analysis

Data were analyzed using repeated measures ANOVA with Bonferroni post-hoc. Analyses were performed using Statistica software (Version 10, Dell, Round Rock, TX) and presented as means ± standard deviations. The alpha was set at p < 0.05.