Effects of an 8-week liquid protein supplementation on resistance training adaptations in untrained healthy college students

Abstract

This study investigates the effects of 8 weeks of liquid protein supplementation on resistance-training adaptations in healthy, untrained college students. Thirty untrained male participants were randomized into two groups: a protein supplement (resistance training exercise (RTE) + protein) and a control (RTE). Both groups underwent resistance training exercises (RTE) three times per week for 8 weeks. The RTE + protein consumed a protein liquid supplement post-exercise, while the RTE consumed water. The results showed a higher degree of change in chest circumference (mean difference = 6.10 cm vs 3.36 cm), maximal bench press strength (mean difference = 16.00 kg vs 8.93 kg, P = 0.007) and maximal squat strength (mean difference = 42.33 kg and 27.32 kg, P = 0.018) in the RTE + protein group compared to the RTE group. Both groups demonstrated increases in thigh circumference, muscle mass, and maximal bench press and deep squat repetitions, but no significant differences were observed between the two groups. These findings suggest that post-exercise protein liquid supplementation can enhance the benefits of RTE on muscle strength and body circumference in young untrained adults. The study highlights the importance of post-exercise protein supplementation for beginners seeking to improve muscle performance, and future research should explore the long-term effects and optimal dosages of protein supplementation in different forms. This trial was registered with ChiCTR under the registration number ChiCTR2300076750.

Introduction

Inadequate muscle mass has been associated with elevated risks of chronic diseases, including cardiovascular disease, type II diabetes, and respiratory disorders (Wolfe, 2006; Aquilani et al., 2014; Booth et al., 2017). Resistance training exercise (RTE) is a well-established approach to promote muscle hypertrophy and strength, thereby improving physical fitness and overall health (Herda et al., 2013; Aquilani et al., 2014). However, the adaptive outcomes of RTE are ultimately determined by the dynamic balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB), a process influenced by both the exercise stimulus and amino acid availability (Tipton, Hamilton & Gallagher, 2018; Lv et al., 2022). Protein intake plays a critical role in shifting this balance toward net muscle accretion by stimulating MPS and supporting post-exercise recovery (Moore et al., 2009; Witard, Bannock & Tipton, 2022). Therefore, combining RTE with protein supplementation represents an effective strategy to enhance muscle mass and strength adaptations.

Beyond promoting anabolic responses, protein supplementation may also help mitigate the adverse effects of exercise-induced muscle damage. Resistance training is known to induce muscle fiber microtrauma, provoke acute inflammatory responses, and cause delayed-onset muscle soreness (DOMS), all of which may transiently impair neuromuscular function and physical performance (Pasiakos, Lieberman & McLellan, 2014). These post-exercise symptoms, such as soreness and fatigue, may reduce exercise adherence by diminishing training motivation and compromising session frequency or quality. A systematic review suggests that peri-exercise protein ingestion can mitigate muscle damage–induced performance decrements and support the remodeling of damaged muscle fibers (Pearson, Hind & Macnaughton, 2023). However, the effectiveness of post-exercise protein supplementation in achieving these outcomes remains relatively inconclusive, with limited evidence available to confirm its benefits.

While most of research has focused on the benefits of protein supplementation for muscle adaptation, emerging evidence also suggests a potential role in skeletal health. Numerous studies have established the efficacy of protein supplementation combined with resistance training in enhancing lean tissue mass and muscle strength in both men and women over 8 weeks (Candow et al., 2006; Taylor et al., 2016). Additionally, resistance training independently promotes bone health by stimulating bone formation and enhancing skeletal integrity (Fujimura et al., 1997; Collados-Gómez et al., 2018; Gómez et al., 2021; Wakolbinger-Habel et al., 2022). Despite these established benefits, relatively few studies have simultaneously assessed the concurrent effects of protein supplementation on muscle hypertrophy and bone metabolism. This gap is particularly evident when evaluating biochemical markers during the initial adaptation phase in resistance-training–naïve individuals (Morton et al., 2018).

To optimize post-exercise muscle protein remodeling and repair, an appropriate protein intake strategy is essential for maximizing MPS. The optimal intake varies depending on factors such as age, training regimen, protein source, and timing of ingestion (Aquilani et al., 2014; Kerksick et al., 2017; Zhou et al., 2024). The effectiveness of protein supplementation is influenced by nutrient timing, as anabolic activity peaks approximately 30 min post-exercise, highlighting the critical importance of rapid nutrient absorption during this period (Ivy & Portman, 2004). A study by Van Wijck et al. (2013) observed that performing resistance exercises such as leg presses and leg extensions led to a 35% elevation in plasma intestinal fatty acid-binding protein (I-FABP) levels within 30 min in recreationally trained males, indicating that such training may temporarily impair gastrointestinal protein digestion and absorption efficiency. In this context, supplement formulation becomes significant, as previous in vitro research indicates that liquid protein formulations exhibit superior gastric digestion characteristics and achieve higher protein hydrolysis rates than powder-based supplements (Xu et al., 2025). For instance, a specific ready-to-drink (RTD) liquid protein product demonstrated approximately 1.5 times greater protein hydrolysis after 30 min of simulated gastric digestion, suggesting enhanced bioavailability. Given the importance of post-exercise nutrient availability and the potential gastrointestinal limitations following resistance training, evaluating practical supplementation strategies becomes crucial. In particular, ready-to-drink protein formulations may offer physiological advantages by enhancing amino acid delivery during the post-exercise anabolic window.

Considering these factors, this study aimed to evaluate the effects of 8 weeks of post-exercise RTD protein supplementation on muscle strength, body composition, and biochemical markers of muscle recovery and bone metabolism in untrained young adults. We hypothesized that post-exercise RTD protein supplementation would lead to greater improvements in muscle strength andmass, and more favorable biochemical adaptations than resistance training alone. The findings may offer valuable insights into the practical nutritional strategies required to optimize muscle and bone adaptations during resistance training.

Materials & Methods

Participants

Thirty healthy male college students aged 18–35 years with a body mass index (BMI) between 18.5 and 23.9 kg/m² and without prior resistance training experience were recruited for this study. This population was selected because untrained young adults exhibit heightened neuromuscular plasticity and anabolic sensitivity, making them an ideal group to observe early adaptations to resistance training and the potential additive effects of protein supplementation. Furthermore, young adult males typically demonstrate more stable hormone levels and dietary patterns, reducing variability in training and nutritional responses. All participants completed a screening process that included a questionnaire, body composition testing, and a non-consecutive 3-day, 24-hour food record (FR) (including two weekdays and one weekend). Exclusion criteria included unwillingness to participate in RTE, allergies to study products, cardiovascular disease, gastrointestinal disorders, thyroid dysfunction, other severe clinical conditions, use of nutritional supplements, heavy smoking, and alcoholism.

This FR method has been widely validated in Chinese adult populations (Shi et al., 2008; Lyu et al., 2014; Song et al., 2017). In particular, three-day food FRs have demonstrated higher correlations and higher agreement proportions of quartile classification with 9-day FRs compared with food frequency questionnaires (FFQs), while maintaining acceptable relative validity for dietary assessment (Yang et al., 2010). Considering the relatively short, 8-week intervention period in this study, the 3-day FRs were considered the most appropriate approach to capture habitual dietary intake with sufficient accuracy and participant compliance.

In this study, we employed a stratified randomization method to ensure balanced distribution of participants across the two groups. Specifically, we used computer-generated random numbers to allocate participants to either the RTE + protein group or the RTE group. The stratification was based on body fat percentage and protein intake (from the 3-day 24 h food diary completed during the screening process), ensuring that these variables were comparable within each stratum. This generated four strata. Within each stratum, participants were randomly assigned to either the intervention or control group in a 1:1 ratio using the runiform function in Stata (version 18.0, StataCorp, College Station, TX, USA), which provided a reliable and unbiased method for generating the random numbers and assigning participants to the respective groups. This approach helped to minimize potential confounding effects and enhance comparability between the two groups.

Participants were recruited through both online and on-campus advertisements. Written informed consent was obtained from all participants prior to enrollment. This study was approved by the Nankai University Biomedical Ethics Review (NKUIRB2022138) and registered at the Chinese Clinical Trial Registry (ChiCTR2300076750).

Training program and intervention protocol

The study was conducted by the Nankai University M-Action Joint Laboratory of Nutrition Research as a randomized controlled trial over 10 weeks for each participant, including a 1-week baseline test phase (week 1), an 8-week RTE phase (weeks 2-9), and a 1-week exit test phase (week 10). Participants from both groups visited the study center for tests and blood sample collection at baseline (week 1) and after 8 weeks of intervention (week 10).

The recruitment period was from March 1st to May 1st, 2023. During the intervention, participants performed 40 min of RTE three times per week under the guidance of a professional trainer. The structured RTE program targeted major skeletal muscle groups of the chest and legs.

Each session followed a full-body training approach, targeting major skeletal muscle groups in both the upper and lower body. The core exercises included: Smith machine bench press, Smith machine deep squat, leg flexion, Smith machine bent-over row, and Smith machine Romanian deadlift. All five exercises were performed in every training session, ensuring consistent stimulation across muscle groups. Each exercise was completed in four sets of 8–12 repetitions, with 60–90 s rest between sets, aimed at enhancing both muscle strength and endurance. The resistance training protocol followed a progressive overload model, with individualized load adjustments made every 1–2 weeks. Training intensity was monitored using the Borg CR-10 scale immediately after each session. Participants were instructed to maintain a rating of perceived exertion (RPE) between 5 and 7, indicating a moderate-to-high intensity. This approach ensured that participants could complete all prescribed sets within the target range of 8–12 repetitions per set while maintaining proper technique under the supervision of trained professionals. All resistance training sessions were conducted under direct supervision by qualified instructors to ensure adherence to the prescribed program and correct exercise execution. Participants were instructed to refrain from engaging in any additional structured resistance or aerobic training beyond the study protocol during the 8-week intervention. This strict control of training and physical activity was implemented to minimize confounding factors and to strengthen the internal validity of the intervention.

Both groups followed the same training program and maintained their habitual diets. The RTE + protein consumed a protein RTD drink within 30 min after RTE under trainer supervision, while the RTE consumed an equal volume of bottled water. All participants kept detailed records of their RTE program, number of sets and repetitions per set, and the start and end times of each exercise session. Researchers and trainers closely monitored these records to ensure optimal training intensity.

Study outcome

At weeks 1 and 10, participants underwent questionnaires, body composition analysis, body circumference measurements, and tests of muscle maximal strength and endurance. Body composition variables, including body weight, body fat mass, and skeletal muscle mass, were measured using bioelectrical impedance analysis (BIA) (InBody 260, Biospace, CA, USA). BIA has been validated for assessing muscle mass using dual-energy X-ray absorptiometry in healthy adults (Ling et al., 2011; McLester et al., 2020; Yi et al., 2022). BIA measurements were performed in the morning under standardized, uniform conditions for all participants, following an overnight fast and with no vigorous physical activity, caffeine, or alcohol permitted within the preceding 12 h. The InBody 260 system provides whole-body and segmental body composition data, including skeletal muscle mass and fat mass for each body region. These data allow for a more detailed assessment of regional muscular adaptations and changes in body composition in response to the intervention.

Bench press and deep squat maximal strength were measured using a one-repetition maximum (1RM) test, involving an incremental warm-up of progressively heavier sets, completing 5–10 repetitions until participants failed to complete a single repetition (Seo et al., 2012). During the 1RM test, a trained professional monitored participants’ performance, rate of perceived exertion (Borg scale), and tested weight, ensuring proper form and adequate rest periods of 2–3 min between sets. The test continued until participants could only complete one full repetition with correct form, and two failed repetitions at the same load were recorded as their 1RM. An endurance test was performed at least 72 h after the 1RM test using 50% of each participant’s maximal bench press or deep squat strength. Participants performed as many repetitions as possible until the Borg scale reached the score of 10. Only repetitions completed with correct technique were counted.

Body circumference measurements, including chest, waist, hip, upper arm, thigh, and shoulder, were performed by trained staff following the standardized procedures outlined by the International Society for the Advancement of Kinanthropometry (ISAK). All measurements were taken on the right side of the body using a non-elastic measuring tape, with participants standing in a relaxed anatomical position.

Overnight fasting venous blood and urine were collected the day after the 1RM muscular strength test to assess metabolic biomarkers, including plasma albumin, glucose, insulin, cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, creatine kinase (CK), lactate dehydrogenase (LDH), and bone metabolism biomarkers (β-isomerized C-terminal telopeptides (β-CTx), precursor peptide to the C-terminal terminus of procollagen type I (PICP), and precursor peptide to the N-terminal of procollagen type I (PINP). Biomarkers were measured using a clinical chemistry analyzer and enzyme-linked immunosorbent assay (Cusabio, Houston, TX, USA).

During the 8-week intervention, participants maintained their habitual diets and kept a non-consecutive 3-day 24-hour dietary diary, including one training day and two non-training days. To enhance data quality, participants were provided with standardized guidance, including visual aids for portion size estimation. Nutrient intakes were calculated using the “China Food Composition Tables Standard Edition (6th Edition/Volume 1)” and “China Food Composition Tables (6th Edition/Volume 2)”. A single trained dietitian performed all dietary data entry and nutrient calculations to ensure consistency across all records.

Study product

The study product was a formulated protein liquid supplement in a 300 mL RTD format provided by Shanghai M-Action Health Technology Co., Ltd. Each bottle contained 25 g of a mixture of casein, whey, and collagen peptide, providing 5 g of branched-chain amino acids. Fat, carbohydrate, and fiber were added to improve palatability, along with 14 vitamins and minerals (Table 1).

Table 1. Nutritional content of the study product.

Nutrient component	Per bottle (300 mL)
Energy	600 kJ
Protein	25.0 g
Fat	0.9 g
Carbohydrate	6.9 g
Dietary Fiber	3.8 g
Calcium	540.0 mg
Sodium	100.0 mg
Phosphorus	318.0 mg
Selenium	10.0 g
Vitamin C	19.5 mg
Vitamin E	2.79 mg-TE
Vitamin D	4.0 g
Vitamin B1	0.29 mg
Vitamin B2	0.29 mg
Vitamin B6	0.29 mg
Vitamin B12	0.60 g
Biotin	6.0 g
Niacin	3.45 mg

Compliance with the intervention

Participants were required to perform 40 min of RTE at a designated gym at the study center. A research assistant documented the date and duration of RTE, intake time of the protein supplement or bottled water, and photos of the completed drinks. Adherence to the intervention was defined as completing at least 90% of the effective exercise sessions. Reasons for non-compliance were documented. If a participant failed to follow the exercise or protein intervention for the first time, the researcher would communicate with the participant face-to-face or by telephone to improve future adherence. Participants who failed to complete more than 10% of the sessions (2 sessions) were asked to withdraw from the study.

Adverse events

Adverse reactions during the trial were documented. In accordance with institutional ethical guidelines, participants were informed that, in the event of a verified study-related severe adverse reaction, they would receive appropriate medical care and reimbursement of medical expenses to ensure that no financial burden would be incurred. If any safety concerns or unexpected injuries arose during the study, the intervention would be discontinued immediately for the affected participant, and appropriate follow-up actions would be taken under medical supervision.

Statistical analysis

The sample size was calculated to detect a significant increase in lean body mass of 5.4% (Joy et al., 2013), with a significance level of 0.05, a power of 0.8, and an attrition rate of 20%, yielding a total of 15 participants in each group. Data entry was performed by two investigators. Only data from participants who completed the full 8-week intervention and post-intervention assessments were included in the final analysis. Participants who withdrew from the study or failed to meet the adherence criteria (≥75% of scheduled training sessions) were excluded from the per-protocol analysis. The collected data were assessed for normality using the Shapiro–Wilk test. Between-group differences for each variable were examined using absolute values and changes from baseline to post-intervention. This study used a stratified statistical strategy for data analysis: based on the data’s normality, we used a paired-samples t-test to compare changes before and after the intervention within each group; for comparisons of changes in each variable during the intervention between the two groups, we used an independent t-test. For categorical variables, chi-square tests were used to assess between-group differences at each time point. All statistical tests were two-tailed. A p-value of ≤ 0.05 was considered statistically significant, while p-values between 0.05 and 0.10 were interpreted as approaching statistically significant (indicative of a trend). All data processing was performed using Stata (version 18.0, StataCorp, College Station, TX, USA).

Results

Participant characteristics and dietary intake

All the participants in the RTE + protein group completed the intervention trial. Fourteen participants in the RTE group completed the trial, and one participant withdrew due to fever; this participant’s data were excluded from further analyses (Fig. 1). Participants were 18–32 years old, with an average age of 21.2 years.

Figure 1. CONSORT diagram showing the progression of participants through the study trial.

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At baseline, the control and protein supplement groups had no significant differences in BMI, body fat percentage, and daily protein intake, and both maintained normal blood indicators. Nutrient intake as recorded in the dietary diary at baseline was comparable in both groups, with similar intakes of energy, carbohydrates, fat, and fiber. During the intervention period, the daily consumption of energy, carbohydrate, fat, and fiber was similar between the two groups. However, the protein supplement group had significantly higher intakes of protein, calcium, and vitamin D. Specifically, the RTE group consumed an average of 1,821.71 ± 467.04 kcal of energy, 237.42 ± 71.49 g of carbohydrate, 64 ± 21.20 g of fat, and 8.39 ± 5.2 g of fiber. The RTE + protein consumed an average of 2,015.82 ± 336.86 kcal of energy, 240.73 ± 49.19 g of carbohydrate, 70.41 ± 21.23 g of fat, and 10.27 ± 3.26 g of fiber. As expected, the RTE + protein had significantly higher daily intakes of protein (97.74 ± 29.36 g, p = 0.05 or 1.51 ± 0.42 g/kg body weight, p = 0.03), calcium (531.44 ± 172.81 mg, p = 0.03), and vitamin D (3.21 ± 1.18 µg, p = 0.02) compared to the RTE (78.97 ± 19.63 g or 1.20 ± 0.27 g/kg body weight for protein, 364.87 ± 223.69 mg for calcium, and 2.01 ± 1.37 µg for vitamin D).

Body circumference

At baseline, there were no significant differences in body circumferences between the two groups. After 8 weeks of RTE, both groups demonstrated significant changes in various body circumferences. Specifically, the average chest circumference, upper arm circumference, and shoulder circumference all increased significantly, while the average hip circumference decreased significantly compared to baseline. Regarding body circumference, both groups demonstrated significant improvements in average chest circumference, with the RTE + protein group showing a greater increase (PΔ = 0.015). Specifically, chest circumference rose from 85.18 ± 3.51 cm to 88.54 ± 4.02 cm in the RTE group, and from 83.67 ± 3.59 cm to 89.77 ±4.28 cm in the RTE + protein group (Table 2).

Table 2. Changes in body circumference before and after 8 weeks of RTE.

		Within-group comparison				Between-group comparison
Body circumference	Group	Pre interventiona	Post interventiona	Cohens_d [95 CI]	P b	Cohens_d [95 CI]	PΔc
Chest (cm)	RTE	85.18 (3.51)	88.54 (4.02)	−1.21 [−1.89, −0.50]	0.001	−0.96 [−1.73, −0.18]	0.015
	RTE + protein	83.67 (3.59)	89.77 (4.28)	−2.10 [−3.02, −1.17]	<0.001
Waist (cm)	RTE	74.50 (3.38)	75.82 (4.90)	−0.40 [−0.93, 0.16]	0.163	0.63 [−0.12, 1.38]	0.100
	RTE + protein	74.93 (4.21)	74.27 (5.10)	0.23 [−0.29, 0.74]	0.394
Hip (cm)	RTE	94.18 (4.20)	91.75 (2.89)	0.74 [0.14, 1.33]	0.016	−0.15 [−0.88, 0.58]	0.686
	RTE + protein	92.23 (3.45)	90.27 (2.24)	0.70 [0.12, 1.26]	0.017
Upper arm (cm)	RTE	26.75 (2.04)	27.61 (1.99)	−0.59 [−1.15, −0.01]	0.047	−0.19 [−0.92, 0.54]	0.618
	RTE + protein	25.93 (1.28)	27.03 (1.20)	−0.98 [−1.59, −0.35]	0.002
Thigh (cm)	RTE	48.46 (3.29)	50.39 (3.48)	−0.78 [−1.37, −0.16]	0.013	−0.73 [−1.48, 0.03]	0.058
	RTE + protein	47.87 (3.12)	51.63 (3.56)	−1.50 [−2.23, −0.74]	<0.001

Notes.

^aData are shown as the mean (standard deviation).

^bWithin-group changes were assessed using paired-sample t-tests, comparing pre- and post-intervention values within each group

^cBetween-group differences in changes during the intervention were assessed using independent t-tests.

Regarding average hip circumference, the RTE group demonstrated a significant decrease from 94.18 ± 4.20 cm to 91.75 ± 2.89 cm, and the RTE + protein also decreased from 92.23 ± 3.45 cm to 90.27 ± 2.24 cm. The decrease in the RTE + protein was slightly smaller than that in the RTE, with this between-group difference approaching statistical statistical significance (PΔ = 0.058).

In terms of limb circumferences, the RTE + protein group showed significant increases in both upper arm (25.93 ± 1.28 cm to 27.03 ± 1.20 cm) and thigh circumference (47.87 ± 3.12 cm to 51.63 ± 3.56 cm). The RTE group demonstrated a significant increase in thigh circumference (48.46 ± 3.29 cm to 50.39  ± 3.48 cm), whereas the increase in upper arm circumference did not reach statistical significance. No significant changes in waist circumference were observed in either group.

Body composition

At baseline, no significant differences in body composition were observed between the two groups (Table 3). After 8 weeks of RTE, both groups showed improvements in multiple body composition indices while maintaining overall body weight and body fat. Specifically, both groups showed increases in lean body mass following the intervention; however, the improvement reached significance only in the RTE + protein group (53.63 ± 4.67 to 54.53 ± 5.34 kg, P = 0.019), whereas the change in the RTE group was not significant (53.79 ± 5.29 to 54.28 ± 4.90 kg, P = 0.155). The between-group difference was not statistically significant (PΔ = 0.275). Similarly, the RTE group demonstrated a non-significant increase (30.24 ± 3.23 to 30.58 ± 3.02 kg, P = 0.099), while the RTE + protein group achieved a significant gain (30.14 ± 2.84 to 30.72 ± 3.25 kg, P = 0.018). Again, the between-group difference was not significant (PΔ = 0.518). Regarding body fat percentage, the RTE + protein group showed a significant reduction (16.29  ± 4.36% to 15.60 ± 4.57%, P = 0.026), whereas the RTE group exhibited only a non-significant decrease (18.81 ±4.29% to 17.67 ± 4.00%, P = 0.101). The between-group comparison was not significant (PΔ = 0.224).

Table 3. Changes in body composition before and after 8 weeks of RTE.

	RTE group (n = 14)				RTE + protein group (n = 15)				Between group comparison
	Pre a	Post a	Cohens_d [95 CI]	P b	Pre a	Post a	Cohens_d [95 CI]	P b	Cohens_d [95 CI]	PΔ c
Age (y)	21.14 (3.42)	21.29 (3.34)	−0.42 [−0.96, 0.14]	0.144	21.20 (3.28)	21.33 (3.27)	−0.38 [−0.90, 0.15]	0.165	0.05 [−0.68, 0.77]	0.444
Height (cm)	173.93 (5.02)	173.93 (5.02)	–	–	174.33 (4.50)	174.33 (4.50)	–	–	–	0.257
Weight (kg)	66.24 (5.42)	65.97 (5.58)	0.16 [−0.37, 0.69]	0.554	64.15 (5.51)	64.67 (5.93)	−0.26 [−0.77, 0.26]	0.327	−0.43 [−1.16, 0.31]	0.279
Body Fat (kg)	12.46 (2.91)	11.69 (2.97)	0.43 [−0.13, 0.97]	0.133	10.52 (3.25)	10.14 (3.39)	−0.38 [−0.90, 0.15]	0.157	−0.27 [−1.00, 0.46]	0.424
Lean Body Mass (kg)	53.79 (5.29)	54.28 (4.90)	−0.40 [−0.94, 0.15]	0.155	53.63 (4.67)	54.53 (5.34)	−0.68 [−1.23, −0.11]	0.019	−0.32 [−1.05, 0.42]	0.275
Skeletal Muscle Mass (kg)	30.24 (3.23)	30.58 (3.02)	−0.48 [−1.02, 0.09]	0.099	30.14 (2.84)	30.72 (3.25)	−0.69 [−1.24, −0.11]	0.018	−0.30 [−1.03, 0.43]	0.518
BMI (kg/m2)	21.90 (1.50)	21.81 (1.55)	0.15 [−0.38, 0.68]	0.581	21.09 (1.41)	21.25 (1.53)	−0.26 [−0.77, 0.26]	0.335	−0.41 [−1.15, 0.33]	0.810
Body Fat Percentage (%)	18.81 (4.29)	17.67 (4.00)	0.47 [−0.09, 1.02]	0.101	16.29 (4.36)	15.60 (4.57)	0.64 [0.08, 1.19]	0.026	−0.24 [−0.97, 0.49]	0.224
Right Upper LMM(kg)	2.81 (0.44)	2.93 (0.46)	−1.07 [−1.73, −0.40]	0.002	2.74 (0.32)	2.87 (0.38)	−0.80 [−1.38, −0.21]	0.007	−0.09 [−0.82, 0.64]	0.491
Left Upper LMM (kg)	2.74 (0.44)	2.83 (0.44)	−0.91 [−1.52, −0.27]	0.005	2.70 (0.32)	2.85 (0.39)	−1.04 [−1.66, −0.39]	0.002	−0.46 [−1.20, 0.28]	0.416
Trunk Muscle Mass (kg)	23.21 (2.47)	23.72 (2.48)	−0.91 [−1.53, −0.27]	0.005	22.91 (1.89)	23.61 (2.21)	−0.81 [−1.38, −0.21]	0.007	−0.26 [−0.99, 0.47]	0.365
Right Lower LMM (kg)	8.73 (0.88)	8.91 (0.87)	−0.81 [−1.41, −0.19]	0.009	8.82 (0.91)	8.93 (0.96)	−0.50 [−1.03, 0.05]	0.075	0.31 [−0.43, 1.04]	0.665
Left Lower LMM (kg)	8.67 (0.88)	8.86 (0.86)	−0.71 [−1.29, −0.11]	0.019	8.81 (0.94)	8.91 (0.96)	−0.43 [−0.95, 0.11]	0.122	0.34 [−0.39, 1.07]	0.200
Right Upper LFM (kg)	0.67 (0.25)	0.59 (0.24)	0.61 [0.03, 1.17]	0.040	0.56 (0.24)	0.49 (0.25)	0.74 [0.16, 1.31]	0.013	−0.16 [−0.89, 0.57]	0.429
Left Upper LFM (kg)	0.70 (0.25)	0.61 (0.24)	0.63 [0.05, 1.20]	0.034	0.56 (0.24)	0.53 (0.25)	0.46 [−0.08, 0.99]	0.096	−0.49 [−1.22, 0.26]	0.737
Trunk Fat Mass (kg)	6.04 (1.62)	5.60 (1.71)	0.42 [−0.13, 0.96]	0.139	4.88 (1.82)	4.69 (1.86)	0.31 [−0.21, 0.83]	0.246	−0.30 [−1.03, 0.44]	0.737
Right Lower LFM (kg)	2.01 (0.40)	1.91 (0.41)	0.38 [−0.17, 0.91]	0.182	1.77 (0.46)	1.71 (0.52)	0.41 [−0.13, 0.93]	0.136	−0.13 [−0.85, 0.60]	0.389
Left Lower LFM (kg)	2.00 (0.39)	1.90 (0.42)	0.39 [−0.16, 0.93]	0.165	1.77 (0.46)	1.70 (0.51)	0.45 [−0.09, 0.98]	0.102	−0.13 [−0.85, 0.60]	0.257

Notes.

^aData are shown as the mean (standard deviation).

^bWithin-group changes were assessed using paired-sample t-tests, comparing pre- and post-intervention values within each group.

^cBetween-group differences in changes during the intervention were assessed using independent t-tests.

Abbreviations

BMI

body mass index

LMM

limb muscle mass

LFM

limb fat mass

In terms of regional muscle mass, both groups demonstrated significant increases in trunk muscle mass (RTE: 23.21 ± 2.47 to 23.72 ± 2.48 kg, P = 0.005; RTE + protein: 22.91 ± 1.89 to 23.61 ± 2.21 kg, P = 0.007). Additionally, the RTE + protein group showed significant improvements in right upper limb muscle mass (2.74 ± 0.32 to 2.87 ± 0.38 kg, P = 0.007) and left upper limb muscle mass (2.70 ± 0.32 to 2.85 ± 0.39 kg, P = 0.002). The RTE group exhibited significant increases in right lower limb (8.73 ± 0.88 to 8.91 ± 0.87 kg, P = 0.009) and left lower limb muscle mass (8.67 ± 0.88 to 8.86 ± 0.86 kg, P = 0.019). However, none of the between-group differences in regional muscle mass reached statistical significance (all PΔ > 0.05).

Overall, within-group analyses indicated that both RTE and RTE + protein interventions significantly improved lean and limb muscle mass, with more consistent effects in the RTE + protein group. Nevertheless, between-group comparisons revealed no statistically significant differences in the magnitude of change between the two interventions.

Muscular maximal strength and endurance

At baseline, no significant differences were observed between the two groups in maximal strength or maximal repetitions for the bench press and deep squat. After 8 weeks of RTE, both groups exhibited significant improvements in muscle strength and endurance (Table 4).

Table 4. Changes in muscle strength and endurance before and after 8 weeks of RTE.

		Within-group comparison				Between-group comparison
	Group	Pre intervention a	Post intervention a	Cohens_d [95 CI]	P b	Cohens_d [95 CI]	Pdelta c
Bench press maximal strength (kg)	RTE	56.61 (17.64)	65.54 (15.51)	−1.12 [−1.79, −0.43]	0.001	−1.07 [−1.85, −0.28]	0.007
	RTE + protein	57.00 (12.26)	73.00 (12.00)	−3.21 [−4.48, −1.92]	<0.001
Bench press maximal repetition number (reps)	RTE	31.29 (9.80)	38.07 (9.17)	−0.58 [−1.14, −0.01]	0.048	0.23 [−0.50, 0.96]	0.540
	RTE + protein	34.13 (8.88)	38.40 (8.88)	−0.42 [−0.94, 0.12]	0.129
Deep squat maximal strength (kg)	RTE	91.25 (17.64)	118.57 (22.91)	−1.42 [−2.16, −0.65]	<0.001	−0.94 [−1.70, −0.16]	0.018
	RTE + protein	93.00 (10.10)	135.33 (17.47)	−3.47 [−4.83, −2.10]	<0.001
Deep squat maximal repetition number (reps)	RTE	36.43 (13.72)	45.14 (13.04)	−0.44 [−0.99, 0.11]	0.120	−0.13 [−0.87, 0.62]	0.739
	RTE + protein	35.13 (7.94)	46.93 (12.89)	−1.04 [−1.68, −0.37]	0.002

Notes.

^aData are shown as the mean (standard deviation).

^bWithin-group changes were assessed using paired-sample t-tests, comparing pre- and post-intervention values within each group.

^cBetween-group differences in changes during the intervention were assessed using independent t-tests.

For maximal strength, the RTE group’s bench press increased from 56.61 ± 17.64 to 65.54 ± 15.51 kg (P = 0.001), whereas the RTE + protein group achieved a larger gain from 57.00 ± 2.26 to 73.00 ± 12.00 kg (P < 0.001). Similarly, deep squat maximal strength rose significantly in both groups, from 91.25 ± 17.64 to 118.57 ± 22.91 kg (P < 0.001) in the RTE group and from 93.00 ± 10.10 to 135.33 ± 17.47 kg (P < 0.001) in the RTE + protein group. Between-group analyses indicated that strength gains in both exercises were significantly greater in the RTE + protein group (bench press, PΔ = 0.007; deep squat, PΔ = 0.018).

For muscular endurance, both groups showed improvements in maximal repetitions. The RTE group increased bench press repetitions from 31.29 ± 9.80 to 38.07 ± 9.17 repetitions (P = 0.048), while the RTE + protein group increased deep squat repetitions from 35.13 ± 7.94 to 46.93 ± 12.89 repetitions (P = 0.002). However, between-group comparisons revealed no significant differences in endurance gains (all PΔ >0.05).

Overall, while both interventions enhanced muscle strength and endurance, the RTE + protein group demonstrated superior improvements in maximal strength for both the bench press and deep squat, suggesting that post-exercise protein supplementation may potentiate strength adaptations to RTE.

Biochemical measurements

As shown in Table 5, plasma CK and LDH levels decreased after 8 weeks of RTE in both groups, but the changes did not reach statistical significance, and no between-group differences were observed. Urinary urobilinogen increased in the RTE group but decreased in the RTE + protein group following training; however, these changes were not significant. Given that plasma CK, LDH, and urinary urobilinogen are established biomarkers of muscle damage and exercise-induced fatigue (Callegari et al., 2017; Cao et al., 2020), these findings suggest that post-exercise protein supplementation had no significant effect on muscle damage or recovery following RTE.

Table 5. Changes in blood biomarkers before and after 8 weeks of RTE.

Variable	Group	Pre intervention	Post intervention	Cohens_d [95 CI]	P b
TBIL (μmol/L)	RTE	14.71 ± 5.73	15.31 ± 4.53	−0.12 [−0.65, 0.40]	0.649
	RTE+Protein	16.39 ± 8.88	16.02 ± 6.92	0.07 [−0.43, 0.58]	0.778
DBIL (μmol/L)	RTE	5.47 ± 2.17	5.34 ± 1.70	0.07 [−0.46, 0.59]	0.806
	RTE+Protein	5.91 ± 2.95	5.41 ± 1.61	0.21 [−0.30, 0.72]	0.423
AST (U/L)	RTE	23.71 ± 4.61	35.57 ± 58.96	−0.20 [−0.73, 0.33]	0.468
	RTE+Protein	27.13 ± 9.93	21.60 ± 5.34	0.55 [−0.01, 1.08]	0.053
S/L	RTE	1.91 ± 0.52	1.71 ± 0.52	0.45 [−0.11, 0.99]	0.119
	RTE+Protein	1.67 ± 0.71	1.55 ± 0.62	0.16 [−0.36, 0.66]	0.553
ALT (U/L)	RTE	13.29 ± 4.32	20.00 ± 25.64	−0.25 [−0.78, 0.28]	0.358
	RTE+Protein	19.87 ± 12.44	16.93 ± 9.87	0.29 [−0.23, 0.80]	0.279
TP (g/L)	RTE	73.46 ± 2.71	73.07 ± 2.76	0.15 [−0.38, 0.67]	0.595
	RTE+Protein	71.80 ± 2.85	71.05 ± 2.15	0.32 [−0.20, 0.83]	0.235
ALB (g/L)	RTE	50.74 ± 2.61	49.57 ± 2.18	0.62 [0.04, 1.19]	0.037
	RTE+Protein	50.14 ± 1.61	49.22 ± 2.19	0.45 [−0.09, 0.97]	0.104
HDL-CH (mmol/L)	RTE	1.42 ± 0.25	1.39 ± 0.25	0.22 [−0.31, 0.75]	0.417
	RTE+Protein	1.45 ± 0.27	1.45 ± 0.30	0.02 [−0.48, 0.53]	0.924
TG (mmol/L)	RTE	0.85 ± 0.37	0.88 ± 0.31	−0.07 [−0.60, 0.45]	0.791
	RTE+Protein	0.71 ± 0.18	0.79 ± 0.29	−0.37 [−0.89, 0.16]	0.175
CHOL (mmol/L)	RTE	4.19 ± 0.55	4.32 ± 0.55	−0.41 [−0.95, 0.14]	0.148
	RTE+Protein	4.07 ± 0.79	4.06 ± 0.76	0.03 [−0.48, 0.53]	0.924
LDL-CH (mmol/L)	RTE	2.61 ± 0.52	2.75 ± 0.56	−0.47 [−1.01, 0.09]	0.103
	RTE+Protein	2.50 ± 0.70	2.44 ± 0.70	0.15 [−0.36, 0.66]	0.572
Glucose (mmol/L)	RTE	3.89 ± 0.54	4.06 ± 0.51	−0.37 [−0.91, 0.18]	0.189
	RTE+Protein	4.06 ± 0.40	3.88 ± 0.61	0.33 [−0.20, 0.84]	0.225
β-CTx (ng/mL)	RTE	0.51 ± 0.24	0.47 ± 0.16	0.27 [−0.26, 0.80]	0.323
	RTE+Protein	0.51 ± 0.17	0.49 ± 0.18	0.34 [−0.18, 0.86]	0.205
CK (U/L)	RTE	262.86 ± 174.89	239.86 ± 110.82	0.11 [−0.41, 0.64]	0.678
	RTE+Protein	404.93 ± 410.81	268.87 ± 167.27	0.37 [−0.16, 0.88]	0.168
PICP (ng/mL)	RTE	0.66 ± 0.33	0.67 ± 0.35	−0.05 [−0.57, 0.48]	0.864
	RTE+Protein	0.73 ± 0.20	0.76 ± 0.23	−0.18 [−0.69, 0.34]	0.502
PINP (ng/mL)	RTE	107.24 ± 52.52	109.41 ± 44.82	−0.11 [−0.63, 0.42]	0.694
	RTE+Protein	99.99 ± 27.83	105.00 ± 30.22	−0.21 [−0.72, 0.30]	0.425

Notes.

^aData are shown as the mean (standard deviation).

^bWithin-group changes were assessed using paired-sample t-tests, comparing pre- and post-intervention values within each group.

Considering the osteogenic effect of RTE and high dietary protein (Mullins & Sinning, 2005), plasma β–CTx, PICP, and PINP were measured as biomarkers of bone metabolism. There were no significant changes in β–CTx, PICP, and PINP, neither between time points nor between groups. These results suggest that neither RTE nor protein supplementation produced detectable changes in bone metabolism within the duration of this study.

Regardingsafety indicators, no adverse events were reported during the intervention. Plasma liver function markers (AST, ALT, and total bilirubin), renal and metabolic indicators, as well as urinary biomarkers (protein, nitrate, ketones, occult blood, white blood cells, etc.) remained within normal ranges. They showed no clinically relevant changes after intervention. These results confirm that both interventions were well tolerated and did not impair hepatic or renal function. (Table 6).

Table 6. Changes in urinary biomarkers before and after 8 weeks of RTE.

Variable	Group	Pre intervention (N)a	P	Post intervention (N)a	P
BLD	RTE	-(14)	-	-(14)	-
	RTE + Protein	-(15)		-(15)
WBC	RTE	+/-(1), -(13)	0.292	+/-(1), -(13)	0.292
	RTE + Protein	-(15)		-(15)
PRO	RTE	+/-(1), -(13)	0.292	+/-(0), -(14)	0.326
	RTE + Protein	-(15)		+/-(1), -(14)
VC	RTE	0 (14)	0.292	0 (13), 5.6 (1)	0.292
	RTE + Protein	0 (15)		0 (15)
URO	RTE	+(1), -(13)	0.96	+(3), -(11)	0.058
	RTE + Protein	+(1), -(14)		+(0), -(15)
SG	RTE
	RTE + Protein
BIL	RTE	-(14)	-	-(14)	-
	RTE + Protein	-(15)		-(15)
NIT	RTE	+(0), -(14)	0.157	+(1), -(13)	0.96
	RTE + Protein	+(2), -(13)		+(1), -(14)
GLU	RTE	-(14)	-	-(14)	-
	RTE + Protein	-(15)		-(15)
KET	RTE	-(14)	-	-(14)	-
	RTE + Protein	-(15)		-(15)

Notes.

^aData are shown as the numbers of cases, Statistical significance level was set at 0.05.

Abbreviation

BLD

Urinary Occult Blood

WBC

Urinary Leukocytes

PRO

Urinary Protein

VC

Ascorbic Acid

URO

Urobilinogen

SG

Urine Specific Gravity

BIL

Bilirubin

NIT

Nitrite

GLU

Glucose

KET

Ketone Bodies

Discussion

Our study demonstrated that an 8-week post-exercise protein liquid supplement increased chest circumference, maximal bench press strength, and maximal deep squat strength to a greater extent than RTE alone in previously untrained young men. Contrary to our expectations, both groups showed increases in thigh circumference, skeletal muscle mass, lean body mass, and muscle endurance after 8 weeks of RTE; however, protein supplementation did not confer significant additive benefits beyond RTE alone. The post-exercise protein liquid supplement did not alter plasma CK or LDH levels compared withwater consumption. Together, our findings indicated that a combination of an 8-week RTE and post-exercise protein supplementation led to an early, rapid increase in muscle strength and a later, slower increase in muscle mass in young novice male adults. Our findings supported the efficacy of post-RTE protein supplementation, demonstrating that young, untrained adults who received protein liquid supplement providing 25 g of protein experienced superior muscle strength gains during the 8-week RTE.

Our findings on body circumference indicated that the protein supplement provided additional benefits in promoting muscle hypertrophy compared with RTE. The RTE program in our study was specific to the chest and thigh muscles. Following RTE, MPS was the primary metabolic driver, and its stimulation was greater in untrained than in trained individuals, resulting in more substantial morphological adaptations over time (Boone et al., 2015; Witard, Bannock & Tipton, 2022). This was why we found increased chest and thigh circumferences in both groups of young novice men. Additionally, ingestion of 20 g of protein was sufficient to stimulate MPS and mitigate MPB after resistance exercise (Moore et al., 2009). Previous studies demonstrated a significantly greater area of type I and type II muscle fibers when 17.5 g or 25 g of protein were supplemented during RTE compared with RTE alone (Andersen et al., 2005; Hartman et al., 2007). These studies supported our results, showing that protein supplementation after RTE let to greater increases in the circumference of exercised muscle.

Our findings of maximal muscle strength indicated that post-exercise protein supplement promoted favorable adaptive responses to RTE. Short-term RTE consistently demonstrated gains in muscular strength, with rapid increases in the early stages of the RTE period due to neural adaptations in untrained adults (Hughes, Ellefsen & Baar, 2018). It remained unclear whether protein supplementation provided an additional boost in strength gains, since previous studies demonstrated conflicting results (Coburn et al., 2006; Joy et al., 2013; Herda et al., 2013; Paoli et al., 2015; Taylor et al., 2016). These studies were conducted in different gender (male or female), different fitness level (untrained, recreational resistance trained, or basketball player), different protein dosage (20 g, 24 g, 48 g), protein type (rice, whey, milk, etc.), and different protein timing (post-exercise, pre-exercise or both) despite the same study duration of 8 weeks. These factors may influence the extend of protein supplementation’s impact on strength adaptation. Additionally, a published meta-analysis demonstrated different effective protein dosages for muscle strength: >1.2 g of protein/kg body weight/d for bench press strength and >1.6 g of protein/kg body weight/d for lower-body strength (Nunes et al., 2022). Our study demonstrated that protein supplementation (1.5 g/kg body weight/d) significantly enhanced maximal muscle strength in both bench press and deep squat. A possible explanation is that more robust MPS stimulation induced by the protein supplementation, thereby facilitating muscle hypertrophy and muscle mass accretion, as evidenced by significantly increased chest circumference and a thigh circumference approaching statistical significance. Collectively, our findings provide strong evidence that post-exercise protein supplementation at a dosage of <1.6 g/kg/d improved lower-body muscle strength development after 8 weeks of RTE. However, when considering body composition, evidence from a larger meta-analysis of 49 studies with 1863 participants demonstrated that no further gains in fat-free mass were observed when total protein intake exceeded 1.62 g/kg/day (Morton et al., 2018). This suggests that in young adults with adequate baseline protein intake, such as our cohort (close to 1.5 g/kg/day as in this study), the marginal benefits of additional protein supplementation on body composition may be limited, even though strength gains were evident.

Adequate protein intake after RTE was necessary for muscle growth. However, our study demonstrated that RTE increased skeletal muscle mass regardless of protein supplementation. This was contrary to our expectation. One explanation could be that both groups had dietary protein intake (1.2 g/kg body weight/d or 1.5 g/kg body weight/d) sufficient to maintain a muscle anabolic state at rest or during recovery from RTE.. Relatively high baseline protein intake and relatively low supplement dosage may contribute to the absence of an additional effect of a protein intervention in combination with RTE. Another explanation isthat muscle strength increases faster than muscle mass in response to RTE (Hughes, Ellefsen & Baar, 2018). Previous studies demonstrated that 12-week protein supplementation during RTE was more effective than RTE alone in promoting lean body mass accretion (Hartman et al., 2007; Hamarsland et al., 2019; Lynch et al., 2020). Therefore, a longer study duration was needed to demonstrate significant improvements in body composition following post-exercise protein supplementation.

RTE disrupted the plasma membrane of muscle fibers, leading to the release of intramuscular proteins into the serum and increased plasma levels of CK and LDH (Rodrigues et al., 2010). Protein consumption was a common nutritional strategy to reduce muscle damage. In our study, plasma CK and LDH levels showed no significant changes within or between groups after the 8-week intervention. However, these results were consistent with several studies showing no significant differences in post-exercise CK concentrations between control and protein-supplementated groups regardless of protein source or dosage (Kim, Lee & Lee, 2017; Saracino et al., 2020; Jacinto et al., 2021; Pearson, Hind & Macnaughton, 2023). Considering inconclusive evidence in the literature, more studies are needed to assess the effect of protein supplementation on exercise-induced muscle damage.

Our study had several limitations. First, the absence of participant blinding may have introduced expectation bias. Secondly, our study relied on self-reported dietary intake, which may introduce recall bias and measurement error despite standardized instructions. Thirdly, although we controlled protein supplement timing, we did not standardize post-exercise carbohydrate intake to improve compliance, which might also influence muscle glycogen repletion and recovery of muscle function (Kerksick et al., 2017). Additionally, we only measured plasma CK and LDH at 24 h after exercise. However, protein supplementation might have enhanced muscle repair and recovery at 72–96 h, since studies demonstrated that whey protein supplementation lowered plasma CK at 72–96 h but not within 24 h (Nieman et al., 2020). Finally, the use of blood biomarkers as surrogate indicators of bone mass. Although these biomarkers are commonly used to assess bone metabolism, they primarily reflect the dynamic processes of bone formation and resorption rather than directly measuring bone mass or its structural integrity. Therefore, the lack of direct bone mass measurement methods, such as dual-energy X-ray absorptiometry (DEXA) or peripheral quantitative computed tomography (pQCT), limits our ability to draw definitive conclusions about the impact of the intervention on bone mass. Future studies should consider incorporating more direct and comprehensive bone mass assessment techniques to understand better the relationship between protein supplementation, RTE, and bone health. To further optimize training and nutritional strategies, additional research is needed to investigate how different protein formulations—such as liquid versus powder—affect muscle adaptation and bone metabolism in human populations undergoing resistance training. Such studies would help clarify whether differences in formulation characteristics influence physiological outcomes related to recovery, anabolism, and skeletal health.

Conclusions

Findings from this study suggest that post-exercise protein liquid supplementation, when combined with an 8-week RTE program in untrained young adults, may enhance maximal muscle strength and could result ingreater increases in chest circumference than RTE alone. Adequate protein intake supports muscle adaptation during resistance training.. These preliminary findings provide a basis for developing exercise and nutrition strategies to improve muscle strength and hypertrophy, but should be interpreted with caution, given the limited sample size and short study duration.

Funding Statement

This study received funding from Shanghai M-Action Health Technology Co. Ltd, Shanghai, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.