Effects of vitamins C and E supplementation combined with 12-week resistance training in older women with sarcopenia: A randomized, double-blind, placebo-controlled trial

Abstract

Background:

Resistance training (RT) is a fundamental sarcopenia treatment, but its efficacy may be enhanced by nutritional strategies. This study investigated whether combining RT with vitamins C and E supplementation yields additive benefits in sarcopenia patients.

Methods:

Sixty older women with sarcopenia (60–75 years) were randomized to an antioxidant supplementation group (AS; 1000 mg/d vitamin C and 335 mg/d vitamin E) or a placebo group (PLA) following the same elastic-band RT program. Muscle mass, muscle strength, physical performance, oxidative stress-related indices (reduced glutathione [GSH] and oxidized glutathione [GSSG], GSH/GSSG ratio, malondialdehyde, and protein carbonyl), and pro-inflammatory factors (interleukin-6 [IL-6] and tumor necrosis factor-alpha) were evaluated at baseline and after the 12-week intervention.

Results:

After 12 weeks, muscle mass, strength, and physical performance significantly increased (P < .05) in both the AS and PLA groups. However, the AS group had higher increases in arm lean mass (Δ = 0.96 vs 0.59 kg; P = .003, d = 0.74), skeletal muscle mass index (Δ = 0.71 vs 0.42 kg/m²; P = .004, d = 0.71), handgrip strength (Δ = 3.66 vs 1.16 kg; P = .047, d = 0.51), and knee extension strength (Δ = 2.28 vs 1.02 kg; P < .001, d = 0.89) than the PLA group. There were no differences in physical performance between the RT conditions over time. Regarding blood parameters, the AS group had increased GSH (P < .001, d = 1.52) and GSH/GSSG ratio (P < .001, d = 1.52), and reduced GSSG (P < .001, d = 0.96) and malondialdehyde (P < .001, d = 1.65) compared to the PLA group. The serum levels of IL-6 and tumor necrosis factor-alpha significantly decreased in the PLA and AS groups, but IL-6 was lower in the AS group than in the PLA group (P < .001, d = 1.16).

Conclusion:

Vitamins C and E supplementation combined with RT for 12 weeks resulted in superior adaptations in muscle mass and strength compared with RT with placebo, and the underlying mechanism could be related to the alleviation of oxidative stress and inflammation.

1. Introduction

Sarcopenia is a condition characterized by decreased skeletal muscle mass, muscle strength, and physical performance, which can lead to a reduced quality of life and an increased risk of frailty, falls, fractures, and even death.^[1] The fundamental measures for the treatment of age-related muscle abnormalities include exercise and nutritional support, and resistance training (RT) is considered the primary strategy for improving sarcopenia.^[2] However, skeletal muscles produce reactive oxygen species (ROS) during contraction and relaxation, especially with intense and long-duration RT, which leads to oxidative stress.^[3] Oxidative stress is a significant factor that contributes to aging and age-related diseases and plays a central role in the various pathogenic mechanisms of sarcopenia.^[4] Although any type of exercise can increase the maximum cellular adaptive capacity to oxidative stress-induced damage, aging could limit this capacity.^[5] Reduced levels of antioxidant enzymes in aging muscles impair their ability to neutralize excess ROS produced during exercise, which may lead to oxidative damage to lipids and proteins in muscle cells.^[6,7] Furthermore, impaired redox signaling is an important cause of chronic inflammation in sarcopenia and induces the expression of pro-inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which can lead to protein degradation and muscle atrophy.^[8]

Emerging evidence suggests that targeted nutritional supplementation may mitigate oxidative stress by augmenting antioxidant defense mechanisms. Among dietary antioxidants, vitamins C and E have demonstrated particular promise in attenuating oxidative damage through free radical scavenging and lipid peroxidation inhibition.^[9,10] However, the interplay between exogenous antioxidant supplementation and exercise-induced adaptations remains contentious. Previous studies in healthy older adults have shown that supplementation with vitamins C and E may exert bidirectional effects on RT outcomes, potentially enhancing or impeding muscular adaptations based on baseline redox status.^[11,12] This dichotomy underscores the importance of precision nutrition strategies, wherein antioxidant supplementation is tailored to address specific deficiencies, thereby optimizing exercise benefits.^[13] Notably, the sarcopenic population exhibits heightened vulnerability to antioxidant inadequacy due to age-related malnutrition and increased oxidative burden.^[14] Several cross-sectional studies have demonstrated positive correlations between circulating levels of vitamins C and E and sarcopenia parameters, such as muscle mass, strength, and physical performance.^[15,16] Despite these findings, the efficacy of vitamins C and E supplementation in enhancing RT outcomes in patients with sarcopenia remains unclear.

Thus, this study aimed to investigate the effects of vitamins C and E supplementation combined with RT in patients with sarcopenia. To control for potential gender-related differences in antioxidant metabolism and muscle protein turnover, this study specifically focused on older women with sarcopenia. The primary outcome was to evaluate changes in muscle mass, muscle strength, and physical performance following a 12-week intervention. The secondary outcome assessed the impact of supplementation on oxidative stress and inflammation-related blood parameters following the training program. We hypothesized that vitamins C and E supplementation would enhance the beneficial effects of RT on muscle adaptation in older women with sarcopenia by improving redox status and inflammation levels.

2. Materials and methods

2.1. Study design and participants

The study employed a 12-week randomized, double-blind, placebo-controlled experiment. Participants were recruited through posters and flyers, and social media in the communities around Jin Qiu Hospital of Liaoning Province. The inclusion criteria were as follows: women, aged 60 to 75 years; diagnosis of sarcopenia based on Asian Working Group for Sarcopenia criteria and confirmed by (1) + (2) or (1) + (3): (1) low muscle mass: Skeletal Muscle Mass Index (SMI) < 5.4 kg/m² as determined via dual-energy X-ray absorptiometry (DXA), (2) low muscle strength: handgrip strength < 18 kg, (3) low physical performance: 5-time chair stand test ≥ 12 seconds or 6 minutes usual gait speed < 1.0 m/s;^[17] no evident diseases, as determined by a comprehensive medical history evaluation conducted under the supervision of 2 specialized clinicians (see File S1, Supplemental Digital Content, https://links.lww.com/MD/P755); no systematic strength training (<1 session per week) in the 6 months prior to the intervention; and no plans to leave the area during the intervention. The exclusion criteria were individuals who were unable to participate in strength training due to severe cardiovascular disease, acute musculoskeletal injuries, uncontrolled hypertension, recent surgical procedures (within 3 months), neurological disorders affecting motor function, or other medical conditions that contraindicate intense physical exercise. Additionally, individuals taking supplements (e.g., free amino acids, protein, vitamin D, omega-3, antioxidants) or medications (e.g., steroids) that could interfere with the intervention were excluded. Investigator’s uncertainty about the willingness or ability of the subject to comply with the protocol requirements was also considered an additional exclusion criterion.

Overall, 60 participants were recruited and randomly assigned in a 1:1 ratio to either the antioxidant supplementation group (AS; n = 30) or the placebo group (PLA; n = 30) by an alphanumeric code assigned to each participant and training group by external staff not involved in the experiment. Both the AS and PLA groups following the same RT program of 3 sessions per week. The week prior to the start of the intervention, participants were familiarized with the study tests and procedures. Prior to and following the 12 weeks of RT and supplementation, muscle mass, strength, physical performance, and serum concentrations of vitamins C and E, reduced glutathione (GSH) and oxidized glutathione (GSSG), GSH/GSSG ratio, lipid peroxidation markers (malondialdehyde [MDA]), protein oxidation markers (protein carbonyl [PCO]), and pro-inflammatory factors (IL-6, TNF-α) were measured. All measurements were assessed by the same researchers at similar times and conditions. Data analysis was conducted by an independent statistician blinded to group assignment. Intervention administrators were likewise blinded to participant allocation. Participants were instructed to keep their daily lifestyles and eating habits unchanged throughout the study.

All participants were comprehensively informed about the study purpose, procedures, possible benefits, risks, and discomforts. All participants and respective guardians provided their written informed consent to participate in this study. The study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki (revised 2013) and adhered to the CONSORT guidelines, as detailed in File S2, Supplemental Digital Content, https://links.lww.com/MD/P755. The study protocol was approved by the Ethics Committee of Shenyang Sports University for Sports Science Experiments (March 29, 2023/Decision number: [2023]13) before initiation.

2.2. Measurements

Identical baseline and post-intervention (after 12 weeks) assessments were conducted in both groups. All evaluations of muscle mass, strength, physical performance, and anthropometry were performed by blinded staff who were not involved in the training procedures. Blood samples for oxidative stress and inflammation analyses were collected by blinded nurses, also not involved in the training. All measurements took place at the Geriatric Medicine Centre of Liaoning Province.

2.2.1. Muscle mass

Total lean mass (i.e., fat-free and bone-free mass), arm lean mass (including deltoid muscles), trunk lean mass (including gluteal muscles), leg lean mass, and SMI were measured using DXA MEDIX DR (Medilink, France). SMI was calculated as the ratio of the appendicular lean mass (sum of lean mass from both arms and legs) to the height (in meters) squared.^[18] DXA measurements were taken in a fasted state, and the participants were scanned from head to toe in a supine position and advised to wear light clothing and remove metal objects. The test–retest reliability of DXA was high (R = 0.95–0.99) as previously described.^[19] Height (accurate to 0.01 cm) and body weight were determined with the DHM-301B Portable Height and Weight Meter (Yaoyi Instrumentation Co., Ltd., Shanghai, China). Body mass index was calculated as weight (in kg) divided by the square of height (in meters).

2.2.2. Muscle strength and physical performance

Muscle strength was assessed using handgrip strength and knee extension strength. Grip strength was measured using a hydraulic hand dynamometer (Jamar, Jipin Time Technology Co., Beijing, China). Three consecutive measurements of dominant hand grip strength were taken with 15-second pause in seated position, and the values were averaged.^[18] The strength of the lower limb was assessed with knee extension strength by using a hand-held dynamometer (MicroFET3, Hogan Health Industries, Inc., UT) on the dominant leg, which procedure has been previously described.^[20] This knee extension strength test has high inter- and intra-rater reliability (intraclass correlation coefficients [ICC] ≥ 0.95 and ICC = 0.948, respectively).^[21] Moreover, the 5-repetition chair stand test, timed up and go test, and the usual 6-meter gait speed were used to assess the participants’ physical performance by using a previously described procedure.^[17] All physical performance tests were measured 3 times, and the results were averaged.

2.2.3. Blood parameters of oxidative stress and inflammation

Two qualified nurses obtained blood samples from an anterior elbow vein into 2 3.5 mL serum vacutainer tube under fasting (≥12 hours) conditions. The samples were allowed to clot and stored at room temperature for 20 minutes, followed by centrifugation at 3000 rpm for 15 minutes to extract serum. The extracted serum was immediately refrigerated at -80°C until their analysis. Serum concentrations of GSH, GSSG, MDA, PCO, IL-6, and TNF-α were measured in duplicate by using commercially available ELISA kits (MLBIO, Shanghai, China) according to the manufacturer’s instructions. The serum samples for vitamin C measurements were mixed with an equal amount of 10% MPA containing 2 mM disodium-EDTA before freezing. The concentrations of vitamins C (ascorbic acid) and E (α-tocopherol) in the serum samples were determined using HPLC with UV detection.^[22,23] The initial collection occurred the week prior to the start of the intervention, and the final collection was performed after the last training session (>48 hours).

2.3. Training procedures

Both groups completed a 12-week, full-body RT program (3 sessions per week). This study utilized elastic bands (TheraBand; Hygenic Corporation, Akron) instead of free weight equipment for strength training in patients with sarcopenia to reduce injury risks. Training sessions took place at the Geriatric Medicine Centre of Liaoning Province. Participants were divided into small groups of <20 people under the supervision of 2 qualified exercise rehabilitators, who were not involved in the measurement of the dependent variable. Each session consisted of 10 minutes of warm-up exercises followed by approximately 50 minutes of elastic band resistance exercises and 10 minutes of cooling-down exercises at the end. The training program includes: elbow flexion and extension; shoulder abduction; chest press; seated rowing; hip extension and abduction; knee extension; standing heel raise; and squat, which were performed in this order. A detailed description of each exercise is presented in File S3, Supplemental Digital Content, https://links.lww.com/MD/P755.

The specific RT protocol and load progression can be found in Figure 1. The 12-week RT program had an undulating periodized profile, specifically 2 of the sessions each week were “moderate” and 1 varied between “heavy” and “light” every second week. Rest between sets was active such as rhythmic swinging of the limbs or some simple dance movements. During the first 10 weeks, the total training volume gradually increased from 1 to 4 sets; however, during the last 2 weeks, the volume gradually decreased from 4 to 2 sets. Furthermore, from the fourth week of the intervention, all exercises were required to be performed as fast as possible in the concentric phase; in the eccentric phase, they were conducted for 2 to 3 seconds. Training loads were determined using the OMNI-Resistance Exercise Scale (OMNI-RES) of perceived exertion adapted for elastic bands; values ranging from 0 to 10 were defined as minimum and maximum perceived effort, respectively. In the week prior to the start of the training, all participants completed 3 familiarization training sessions. During the sessions, participants performed 2 different exercises, high intensity and low intensity, to determine the range of values for perceived exertion to help them better understand the anchor points of 0 to 10. Light intensity corresponds to 4 to 5 on the OMNI-RES scale, moderate intensity corresponds to 6 to 7, and heavy intensity corresponds to 8 to 9.^[24] Exercise load progressed weekly based on individual improvement. Resistance was increased (via grip width and band color) when exercises were performed correctly without significant fatigue, ensuring intensity remained in the target range. Throughout the training period, exercise intensity was progressively increased by adjusting the elastic band resistance (based on the TheraBand force-elongation table) from yellow to red and further to black. All workout information was confirmed in the training diary. The RT program was based on previous literature,^[25,26] modified to suit the characteristics of the study population, and followed the American College of Sports Medicine guidelines for older adults.^[27]

Figure 1.

Resistance training protocol schematic. OMNI-RES = OMNI-Resistance Exercise Scale.

2.4. Supplementation protocol

The supplementation protocol was based on previous literature.^[11] Participants in the AS group were supplemented with 1000 mg of vitamin C and 335 mg of vitamin E per day (Sanofi-Aventis Healthcare Pty Ltd, Australia). The following supplementations were administered: on training days: 1 tablet of vitamin C (500 mg ascorbic acid) 1 to 3 hours before training, 1 tablet of vitamin E (335 mg DL-α-tocopherol acetate), and vitamin C in the hour after training; on non-training days: 1 tablet of vitamin C in the morning, 1 tablet of vitamin E and vitamin C in the evening. Thus, the daily dosage was 1000 mg of vitamin C and 335 mg vitamin E. This supplementary form has been reported to increase the bioavailability of vitamins and ensure that antioxidant levels in the blood remain high when oxidative stress is at its highest.^[25] Participants in the PLA group were supplemented with placebo (lactose) in the same way and time each day; the placebo tablets had the same shape and appearance as the vitamin tablets but had no vitamin or antioxidant content. Participants were provided with a week’s ration of supplements by using dispenser pill boxes; at the end of the last training session of each week, the new week’s supplements were distributed, and the empty boxes were collected to ensure compliance.

2.5. Dietary and activity control

Dietary intake was assessed using 3-day food records (2 weekdays and 1 weekend) at baseline. The intake of each food ingredient was analyzed using the Chinese Food Composition Table to estimate total energy consumption; the amounts of proteins, vitamin C, and vitamin E consumed were also calculated.^[28] Physical activity was evaluated using the short-form self-assessment International Physical Activity Questionnaire, which determined the intensity of the usual physical activities.^[29] Participants were instructed to maintain their usual dietary and physical activity habits throughout the study. They were asked to refrain from taking any nutritional supplements other than those provided in the study. Additionally, consumption of antioxidant-rich juices (e.g., grape juice) was to be avoided, and daily intake was limited to no more than 2 glasses of juice and 4 cups of coffee or tea. All relevant information was confirmed in the training diary to ensure strict compliance.

2.6. Statistical analysis

A priori sample size estimation was performed using G*Power software (Version 3.1.9.7, Heinrich Heine University, Düsseldorf, Germany). Based on a previous study,^[25] with a significance level (α) of 0.05, a power (1-β) of 0.80, and an expected mean group difference of 0.7 kg in muscle mass (the primary outcome), a minimum of 16 participants per group was required. To account for potential attrition, 60 participants were recruited and randomly assigned to either the intervention or control group.

All analyses were conducted on an intention-to-treat basis, including all 60 randomly assigned participants (no missing data). The normality of data distribution was assessed using the Kolmogorov–Smirnov test, and homogeneity of variance was evaluated using Levene test. The results showed that all measured variables followed a normal or approximately normal distribution; therefore, parametric tests were utilized for data analysis. An independent t test was applied to identify any significant difference between 2 groups before the intervention to ensure the comparability. To evaluate the effects of the intervention, a 2-way repeated measures ANOVA was conducted to assess within-group (time effect), between group (group effect), and time–group interaction effects. Effect sizes for interaction effects were reported as partial eta squared (ƞ_p²), with values interpreted as follows: 0.01 < ƞ_p² < 0.06 (small), 0.06 ≤ ƞ_p² ≤ 0.14 (medium), and ƞ_p² > 0.14 (large). If a significant interaction was observed, simple main effects were analyzed using paired samples t test for time comparisons for each group and independent samples t test for group comparisons at each time point. If no significant interaction was detected, the main effects of time and group were analyzed separately using paired t tests and independent t tests, respectively. Effect sizes for main effects and simple main effects were calculated as Cohen d, where 0.80 is considered large, 0.50 is considered medium, and 0.20 is considered small.^[30] The percentage of change of each variable was calculated using the following formula: Δ% = [(post-test score - pretest score)/pretest score] × 100. Statistical significance was set at P < .05. All analyses were performed using SPSS (version 25.0, IBM, Chicago). Graphpad Prism (version 10.4.1, GraphPad Software, San Diego ) was used to generate figures.

3. Results

3.1. Participant characteristics

A total of 60 women with sarcopenia were recruited and randomly allocated to the AS group (n = 30) and the PLA group (n = 30), with no dropouts occurring in either group during the entire intervention period, as shown in Figure 2. The interventions were implemented with close monitoring of participants’ responses, and no adverse events were reported. All participants demonstrated 100% adherence to both the training and supplementation protocols. Baseline assessments revealed that all participants (60/60) had inadequate vitamin C levels (<50 μmol/L), while 57% (34/60) had inadequate vitamin E levels (<20 μmol/L), according to the most commonly used reference values.^[31,32] The baseline characteristics of the participants are shown in Table 1. No significant differences existed between the AS and PLA groups in age, height, weight, body mass index, International Physical Activity Questionnaire score, and dietary intake at the beginning of the study. Figure 3 depicts the percentage changes in measured variables before and after intervention.

Figure 2.

Participants flowchart.

Table 1.

Demographic information and characteristics.

Variable	AS group (n = 30)	PLA group (n = 30)	P value
Age (yr)	66.07 ± 4.05	65.65 ± 4.60	.706
Height (m)	1.64 ± 0.06	1.65 ± 0.07	.426
Weight (kg)	61.03 ± 7.64	61.45 ± 7.46	.830
BMI (kg/m2)	22.55 ± 1.66	22.40 ± 1.67	.735
IPAQ score	452 ± 80	418 ± 65	.619
Dietary intake
Protein intake (g/day)	69 ± 21	75 ± 29	.671
Energy intake (kcal/day)	1898 ± 524	1943 ± 489	.484
Vitamin C intake (mg/day)	78 ± 35	75 ± 32	.750
Vitamin E intake (mg/day)	12 ± 2	12 ± 3	.904

Data are presented as (mean ± SD).

AS = antioxidant supplementation group, BMI = body mass index, IPAQ = International Physical Activity Questionnaire, n = number of participants, PLA = placebo group, SD = standard deviation.

Figure 3.

Changes in muscle mass, muscle strength, physical performance, and blood parameters from baseline. Data are presented as mean with 95% CI. AS = antioxidant supplementation group, GSH = reduced glutathione, GSSG = oxidized glutathione, IL-6 = interleukin-6, MDA = malondialdehyde, PCO = protein carbonyl, PLA = placebo group, SMI = skeletal muscle mass index, TNF-α = tumor necrosis factor-alpha.

3.2. Primary outcomes

Descriptive and inferential analyses of muscle mass, muscle strength, and physical performance are presented in Table 2. Nonsignificant between group differences (P > .05) were found for all variables in the preintervention measurements.

Table 2.

Intervention effects on the muscle mass, strength and physical performance of each group.

Variable)		AS group (n = 30)	Change mean (95% CI)	P value (time)	PLA group (n = 30)	Change mean (95% CI)	P value (time)	P value (group)
Total lean mass (kg	Baseline	34.06 ± 3.15	1.93 (1.24,2.62)	<.001	33.59 ± 3.65	1.50 (0.56, 2.44)	.003	.598
	Week 12	35.99 ± 3.68			35.10 ± 4.08			.375
Arm lean mass (kg)	Baseline	3.40 ± 0.43	0.96 (0.80, 1.12)	<.001	3.31 ± 0.47	0.59 (0.43, 0.75)	<.001	.415
	Week 12	4.36 ± 0.60			3.90 ± 0.56			.003
Leg lean mass (kg)	Baseline	10.51 ± 1.14	0.91 (0.65, 1.17)	<.001	10.92 ± 1.16	0.48 (0.23, 0.74)	.001	.181
	Week 12	11.43 ± 1.44			11.40 ± 1.29			.936
Trunk lean mass (kg)	Baseline	16.88 ± 2.14	0.17 (-0.37, 0.71)	.527	16.03 ± 2.40	0.65 (-0.09, 1.39)	.084	.150
	Week 12	17.05 ± 2.08			16.68 ± 2.44			.528
SMI (kg/m²)	Baseline	5.15 ± 0.19	0.71 (0.59, 0.83)	<.001	5.17 ± 0.20	0.42 (0.30, 0.54)	<.001	.724
	Week 12	5.86 ± 0.37			5.59 ± 0.34			.004
Handgrip strength (kg)	Baseline	16.21 ± 3.89	3.66 (3.31, 4.02)	<.001	16.73 ± 3.97	1.16 (0.64, 1.68)	<.001	.607
	Week 12	19.87 ± 3.64			17.89 ± 3.98			.047
Knee extension strength (kg)	Baseline	13.01 ± 2.90	2.28 (1.56, 3.00)	<.001	12.77 ± 2.92	1.02 (0.11, 1.92)	.030	.744
	Week 12	15.29 ± 1.73			13.78 ± 1.31			<.001
Five-repetition chair stand (s)	Baseline	11.19 ± 2.30	-1.74 (-2.23, -1.25)	<.001	10.98 ± 3.06	-0.34 (-0.66, -0.01)	.041	.760
	Week 12	9.45 ± 1.82			10.65 ± 2.64			.100
Timed up and go test (s)	Baseline	7.88 ± 1.05	-0.36 (-0.49, -0.23)	<.001	7.72 ± 1.01	-0.19 (-0.28, -0.11)	<.001	.548
	Week 12	7.52 ± 0.97			7.53 ± 0.98			.966
6-meter gait speed (s)	Baseline	6.16 ± 1.33	-0.45 (-0.84, -0.05)	.028	5.91 ± 1.58	-0.28 (-0.68, 0.12)	.164	.509
	Week 12	5.71 ± 0.97			5.63 ± 1.07			.749

Data are presented as (mean ± SD).

Note: Significant P-values are highlighted in bold font.

95% CI = 95 % confidence interval, AS = antioxidant supplementation, n = number of participants, PLA = placebo group, SD = standard deviation, SMI = Skeletal Muscle Mass Index.

3.2.1. Effects of the intervention on muscle mass

A significant interaction between group and time was observed for arm lean mass (F = 11.42, P = .001, η_p² = 0.16) and leg lean mass (F = 5.77, P = .019, η_p² = 0.09). After the 12-week intervention, arm lean mass increased in the AS group (P < .001, effect size Cohen {d} = 2.13) and the PLA group (P < .001, d = 1.31) compared with the pretest values. The arm lean mass in the AS group was higher than that in the PLA group in the post-test (P = .003, d = 0.74) (Fig. 4A). The leg lean mass also substantially increased in the AS (P < .001, d = 0.79) and PLA (P = .001, d = 0.41) groups after 12 weeks, while no significant difference was observed between the groups in the post-test (P = .936; d = 0.02) (Fig. 4B).

Figure 4.

Comparison of muscle mass between groups following 12-week intervention. Data are presented as median with interquartile range. **P < .01 versus placebo group; NS indicates not significant. AS = antioxidant supplementation group, PLA = placebo group, SMI = skeletal muscle mass index.

No significant interaction effects were observed for trunk lean mass (F = 1.13, P = .292, η_p² = 0.02) and total lean mass (F = 0.56, P = .458, η_p² = 0.01). The main effects of time (F = 3.28, P = .075, η_p² = 0.05) and group (F = 1.29, P = .260, η_p² = 0.02) were not significant in trunk lean mass (Fig. 4C). In terms of total lean mass, a significant main effect of time (F = 35.86, P < .001, η_p² = 0.38) was observed, which increased over time for the AS (P < .001; d = 0.57) and PLA (P = .003; d = 0.45) groups. However, the main effect of group (F = 0.58, P = .451, η_p² = 0.01) was not significant for total lean mass (Fig. 4D).

A significant interaction between group and time was observed for SMI (F = 12.45, P = .001, η_p² = 0.17). SMI significantly increased over time for the AS (P < .001, d = 3.74) and PLA (P < .001, d = 2.21) groups. Indeed, there was a difference between the groups in the post-test so much so that the SMI in the AS group was better than in the PLA group (P = .004, d = 0.71) (Fig. 4E).

3.2.2. Effects of the intervention on muscle strength and physical performance

A significant interaction between group and time was observed for handgrip strength (F = 65.12, P < .001, η_p² = 0.53). After the 12-week intervention, grip strength increased in the AS (P < .001, d = 0.94) and PLA (P < .001, d = 0.30) groups compared with the pretest values. Intergroup changes showed that grip strength was higher in the AS group than in the PLA group in the post-test (P = .047, d = 0.51) (Fig. 5A).

Figure 5.

Comparison of muscle strength and physical performance between groups following 12-week intervention. Data are presented as median with interquartile range. *P < .05, ***P < .001 versus placebo group; NS indicates not significant. AS = antioxidant supplementation group, PLA = placebo group.

A significant interaction between group and time was observed for knee extension strength (F = 4.90, P = .031, η_p² = 0.08) and 5-repetition chair stand test (F = 24.36, P < .001, η_p² = 0.29). Knee extension strength significantly increased over time in the AS (P < .001; d = 0.79) and PLA (P = .030, d = 0.35) groups. It was higher in the AS group than in the PLA group in the post-test (P < .001, d = 0.89) (Fig. 5B). Regarding 5-repetition chair stand test, intragroup changes decreased in the AS and PLA groups compared with the pretest values (P < .001, d = 0.65 and P = .041, d = 0.12, respectively). No significant difference was observed between the groups in the post-test (P = .100, d = 0.52) (Fig. 5C).

A significant interaction between group and time was observed for timed up and go test (F = 4.98, P = .029, η_p² = 0.08). A significant decrease in timed up and go test was observed over time for the AS (P < .001; d = 0.35) and PLA (P < .001, d = 0.19) groups. No significant difference was observed between the groups in the post-test (P = .966, d = 0.01) (Fig. 5D).

No significant interaction effects were observed for 6-meter gait speed (F = 0.36, P = .551, η_p² = 0.01). A significant main effect of time was found (F = 6.97, P = .011, η_p² = 0.11), with a decrease over time for the AS group (P = .028; d = 0.31) but not for the PLA group (P = .164; d = 0.19). Additionally, the main effect of group (F = 0.32, P = .571, η_p² = 0.01) was not significant for the 6-meter gait speed (Fig. 5E).

3.3. Secondary outcomes

Descriptive and inferential analyses of blood parameters are presented in Table 3. Nonsignificant between-group differences (P > .05) were found for all variables in the preintervention measurements.

Table 3.

Intervention effects on the blood parameters of each group.

Variable		AS group (n = 30)	Change mean (95% CI)	P value (time)	PLA group (n = 30)	Change mean (95% CI)	P value (time)	P value (group)
Vitamin C (µmol/L)	Baseline	32.47 ± 10.59	27.59 (24.38, 30.81)	<.001	29.89 ± 8.53	-2.17 (-4.54, 0.20)	.071	.300
	Week 12	60.06 ± 11.93			27.72 ± 7.47			<.001
Vitamin E (µmol/L)	Baseline	18.46 ± 3.64	11.41 (9.44, 13.37)	<.001	20.32 ± 4.55	-0.88 (-2.44, 0.67)	.254	.084
	Week 12	29.86 ± 4.44			19.43 ± 2.70			<.001
GSH (µmol/L)	Baseline	21.74 ± 2.36	5.16 (4.19, 6.13)	<.001	20.85 ± 2.58	-0.70 (-1.47, 0.06)	.071	.165
	Week 12	26.90 ± 2.90			20.14 ± 2.85			<.001
GSSG (µmol/L)	Baseline	2.47 ± 0.25	0.15 (0.03, 0.27)	.019	2.37 ± 0.39	0.76 (0.57, 0.95)	<.001	.263
	Week 12	2.62 ± 0.29			3.13 ± 0.59			<.001
GSH/GSSG ratio	Baseline	8.90 ± 1.29	1.48 (0.84, 2.12)	<.001	9.07 ± 2.02	-2.39 (-3.02, -1.76)	<.001	.696
	Week 12	10.38 ± 1.48			6.68 ± 1.67			<.001
MDA (µmol/L)	Baseline	7.20 ± 0.62	-1.63 (-1.95, -1.31)	<.001	7.05 ± 0.64	0.66 (0.43, 0.90)	<.001	.354
	Week 12	5.57 ± 0.69			7.72 ± 0.78			<.001
PCO (µmol/L)	Baseline	8.44 ± 0.62	0.10 (-0.32, 0.52)	.607	8.12 ± 0.68	0.78 (0.44, 1.11)	<.001	.061
	Week 12	8.54 ± 0.87			8.89 ± 0.61			.074
IL-6 (pg/mL)	Baseline	42.12 ± 4.36	-10.94 (-12.80, -9.07)	<.001	44.21 ± 4.95	-4.74 (-6.66, -2.82)	<.001	.086
	Week 12	31.18 ± 5.98			39.47 ± 5.74			<.001
TNF-α (pg/mL)	Baseline	81.71 ± 8.47	-24.04 (-25.62, -22.46)	<.001	82.75 ± 12.50	-21.29 (-25.18, -17.41)	<.001	.705
	Week 12	57.67 ± 8.86			61.46 ± 7.81			.082

Data are presented as (mean ± SD).

Note: Significant P-values are highlighted in bold font.

95% CI = 95 % confidence interval, AS = antioxidant supplementation group, GSH = reduced glutathione, GSSG = oxidized glutathione, IL-6 = interleukin-6, MDA = malondialdehyde, n = number of participants, PCO = protein carbonyl, PLA = placebo group, SD = standard deviation, TNF-α = tumor necrosis factor-alpha.

A significant interaction between group and time was observed for vitamin C (F = 234.67, P < .001, η_p² = 0.80) and vitamin E (F = 101.23, P < .001, η_p² = 0.63). After 12 weeks of supplementation, serum concentrations of vitamins C and E were elevated in AS group (P < .001, d = 2.87 and P < .001, d = 2.71, respectively), but not in PLA group (P = .071, d = 0.23 and P = .254, d = 0.22, respectively). In the post-test, both vitamins C and E levels were higher in AS group than in PLA group (P < .001, d = 1.70 and P < .001, d = 1.63, respectively) (Fig. 6A and B).

Figure 6.

Comparison of blood parameters between groups following 12-week intervention. Data are presented as median with interquartile range. ***P < .001 versus placebo group; NS indicates not significant. AS = antioxidant supplementation group, GSH = reduced glutathione, GSSG = oxidized glutathione, IL-6 = interleukin-6, MDA = malondialdehyde, PCO = protein carbonyl, PLA = placebo group, TNF-α = tumor necrosis factor-alpha.

Regarding antioxidant defenses, a significant interaction between group and time was observed for GSH (F = 94.77, P < .001, η_p² = 0.62) and GSSG (F = 29.37, P < .001, η_p² = 0.33). After the 12-week intervention, serum concentrations of GSH increased in the AS group (P < .001, d = 2.07) decreased in the PLA group (P = .071, d = 0.29) compared with the values in the pretest. Indeed, serum concentrations of GSH was higher in the AS group than in the PLA group in the post-test (P < .001, d = 1.52) (Fig. 6C). A significant increase in serum concentrations of GSSG was observed over time for the AS group (P = .019, d = 0.45) and PLA group (P < .001, d = 2.30). Nevertheless, intergroup changes showed that serum concentrations of GSSG was lower in the AS group than the PLA group in the post-test (P < .001, d = 0.96) (Fig. 6D).

In terms of GSH/GSSG ratio, a significant interaction between group and time was observed (F = 74.14, P < .001, η_p² = 0.56). A significant increase in GSH/GSSG ratio was observed over time in the AS group (P < .001, d = 0.88), and a significant decrease was recorded in the PLA group (P < .001, d = 1.41). Intergroup changes showed that GSH/GSSG ratio was higher in the AS group than in the PLA group in the post-test (P < .001, d = 1.52) (Fig. 6E).

Regarding the oxidative damage indices in serum, a significant interaction between group and time was observed for MDA (F = 140.28, P < .001, η_p² = 0.70). A significant decrease in MDA was observed over time in the AS group (P < .001, d = 2.59) and a significant increase in the PLA group (P < .001, d = 1.06). Intergroup changes showed that MDA was lower in the AS group than in the PLA group in the post-test (P < .001, d = 1.65) (Fig. 6F). No significant interaction effects were observed for PCO (F = 6.59, P = .013, η_p² = 0.10). A significant main effect of time was found (F = 11.25, P = .001, η_p² = 0.16), with PCO increasing over time for the PLA group (P < .001; d = 1.15), but not AS group (P = .607; d = 0.15). Additionally, the main effect of group (F = 0.02, P = .896, η_p² < 0.01) was not significant for PCO (Fig. 6G).

In relation to pro-inflammatory factors in serum, a significant interaction between group and time were observed for IL-6 (F = 22.31, P < .001, η_p² = 0.27). A significant decrease in IL-6 was observed over time in the AS (P < .001, d = 2.30) and PLA (P < .001, d = 1.00) groups. Intergroup changes showed that serum IL-6 concentration was lower in the AS group than in the PLA group in the post-test (P < .001, d = 1.16) (Fig. 6H). No significant interaction effects were observed for TNF-α (F = 1.75, P = .191, η_p² = 0.03). A significant main effect of time was found (F = 476.21, P < .001, η_p² = 0.89), with serum TNF-α concentration decreasing over time in the AS (P < .001; d = 2.26) and PLA (P < .001; d = 2.00) groups. However, the main effect of group (F = 1.17, P = .283, η_p² = 0.02) was not significant for TNF-α (Fig. 6I).

4. Discussion

To our knowledge, this is the first study investigating the effects of a 12-week RT combined with vitamins C and E supplementation in older women with sarcopenia. The results partially supported our hypotheses: as most participants had inadequate baseline levels of vitamins C and E, the supplementation group showed significantly greater improvements in arm lean mass, SMI, and upper/lower limb muscle strength compared to the PLA group. However, no significant effects were observed on physical performance tests. Additionally, vitamins C and E supplementation significantly elevated the GSH/GSSG ratio while reducing serum MDA and IL-6 levels.

Both RT conditions significantly increased muscle mass (1.93 kg and 1.50 kg). Although no significant group differences were observed, the results were superior to previous similar studies. Labonte et al^[12] found that 24 weeks of RT combined with vitamins C and E resulted in only a 1.5 kg increase in fat-free mass in healthy older men. The greater increase in muscle mass observed in our study over a shorter intervention period possibly because we only included patients with sarcopenia who had lower muscle mass. Additionally, gender differences may play a role, as studies suggest that combined exercise and nutrition interventions are more effective for elderly women than for men.^[33] In the current study, the increase in muscle mass was mostly apparent in the appendicular lean mass, consistent with previous research on elastic resistance exercise in older women.^[34] The AS group demonstrated greater improvements in arm (28.2% vs 17.8%) and leg lean mass (8.7% vs 4.4%) compared to the PLA group, with significant group differences observed in arm lean mass post-intervention. Appendicular lean mass improvement is crucial in sarcopenia management, as it directly correlates with muscle function maintenance and reduced fall risk.^[1] Furthermore, the AS group showed a greater increase in SMI compared to the PLA group (d = 0.71). Post-test results revealed that 97% of AS participants (29 of 30) achieved an SMI ≥ 5.4 kg/m² (the Asian Working Group for Sarcopenia cutoff for women), compared to only 73% in the PLA group (22 of 30). A recent meta-analysis reported a mean difference of 0.24 kg/m² in improvement effect of RT plus protein supplementation on SMI in sarcopenia compared to RT alone.^[35] Another work observed that a 12-week RT combined with creatine supplementation only increased SMI in older adults by 0.36 kg/m².^[36] In contrast, the current study observed improvements of 0.27 kg/m² and 0.71 kg/m², respectively. Given the considerable evidence that protein and creatine are ideal choices for supporting RT in sarcopenia management,^[37] the combination of vitamins C and E with RT represents a promising and effective intervention for enhancing muscle mass.

Both interventions significantly improved muscle strength, but significantly better results for the AS group compared to the PLA group in the post-intervention handgrip strength (d = 0.51) and knee extension strength (d = 0.89). A handgrip strength increase of 3.66 kg was observed in the AS group, which was superior to the 1.43 kg increase in older women after 20 weeks of elastic band-based RT.^[26] Lu et al^[38] found that RT was effective in enhancing knee extension strength in older people with sarcopenia by a standardized mean difference of 1.36 kg (95% CI: 0.71–2.02). In our study, the PLA group showed a similar increase of 1.02 kg (95% CI: 0.11–1.92), while the AS group exhibited a greater increase of 2.28 kg (95% CI: 1.56–3.00). In predicting health outcomes, low muscle strength is a more meaningful component of sarcopenia definition than muscle mass loss and is independently and significantly associated with an increased risk of all-cause mortality in older adults.^[39] Therefore, based on our results, applying a 12-week RT program combined with vitamins C and E supplementation proved effective in enhancing muscle strength in patients with sarcopenia.

Both interventions significantly improved physical performance at 12 weeks, however, no significant group differences were observed. Consistent with prior research, the observed strength gains were not associated with improvements in most components of physical performance.^[40,41] The improvements in the timed up and go test were similar in both groups (decrease of 3–5 %) but lower than the values in previous studies that used elastic band strength training in older women (decrease of 13–15 %).^[26,42] This discrepancy may be attributed to the slow velocity of movement of our RT program until the fourth week, when the intervention began to require a faster velocity, which is essential for improving physical performance.^[43] Nevertheless, we observed a 15.5% reduction in the 5-repetition chair stand test in the AS group, compared to only 3.1% in the PLA group. This result seems to be clinically significant, as a 10% reduction in time per year is considered the target of interventions to enhance mobility and prevent incident disability.^[44] Additionally, 6-meter gait speed improved only in the AS group, a critical factor associated with enhanced survival.^[45] Although no significant group differences were observed, the AS group outperformed the PLA group in all tests. This may be due to the short intervention period in our study, where improvements in muscle mass and strength were not apparent in response to physical performance, suggesting that extending the intervention duration may reveal groups differences.

After 12 weeks of supplementation, mean serum vitamin C (32.47 ± 10.59–60.06 ± 11.93 μmol/L) and vitamin E (18.46 ± 3.64–29.86 ± 4.44 μmol/L) levels were adequate in the AS group, but not in the PLA group. This suggests that the observed improvements in muscle mass and strength under the 2 RT conditions may be primarily attributed to the elevated levels of antioxidant vitamins in the participants. These findings contrast with previous studies, which reported that supplementation with vitamin C (1000 mg/d) and vitamin E (235 mg/d) attenuated the beneficial effects of 12-week RT on total lean mass and rectus femoris muscle thickness in older adults;^[11] in addition, the improved effect of RT on bone mineral density was impaired.^[46] Notably, in those studies, baseline circulating levels of vitamins C and E were already well above adequate levels. Excessive antioxidant supplementation under such conditions could hinder the normal physiological effects of ROS and exercise-induced adaptive changes.^[47,48] By contrast, most participants in our study had inadequate levels of vitamins C and E. These results support our hypothesis that patients with sarcopenia exhibit poor antioxidant status and may benefit from antioxidant vitamins supplementation, particularly during more intensive and prolonged RT interventions.

Changes in blood biomarkers proved the beneficial effects of vitamins C and E supplementation on physiological adaptation to RT. The GSH/GSSG ratio, a key indicator of exercise-induced oxidative damage risk, typically shows a decrease in GSH and an increase in GSSG following acute exercise, reflecting elevated oxidative stress.^[49] However, regular training has been shown to enhance the adaptive response to oxidative stress. For instance, a previous study found that 12 weeks of RT improved the adaptive response to exercise-induced oxidative stress in older adults.^[50] Regardless of exercise type or intensity, regular exercise exerts antioxidant effects by reducing pro-oxidant markers.^[51] In the present study, we did not observe antioxidant effects from 12 weeks of regular RT, as the GSH/GSSG ratio significantly decreased in the PLA group. In contrast, supplementation with vitamins C and E during RT led to significant increases in serum GSH concentrations and the GSH/GSSG ratio, with values higher than those in the PLA group at 12 weeks (both, d = 1.52). This increase in GSH levels may be attributed to the synergistic effects of vitamins C and E, which promote GSH synthesis, regeneration, and reduced consumption.^[52] Elevated serum GSH levels are particularly significant, as skeletal muscles can deliver GSH to the circulation, and increased serum GSH reflects positive muscle adaptations to exercise training.^[53] Additionally, the PLA group exhibited significant increases in MDA and PCO levels, indicating that even with regular RT, women with sarcopenia and poor antioxidant vitamin status remain at higher risk of oxidative metabolic stress and damage. Vitamins C and E supplementation may counteract MDA by stabilizing myocyte membranes,^[52] as evidenced by significantly lower MDA levels in the AS group compared to the PLA group (d = 1.65). Improvements in MDA levels have been linked to positive adaptive muscle responses to RT in older adults.^[54] Although no significant group differences were observed in PCO levels, vitamins C and E supplementation prevented the increase in PCO (0.10 vs 0.78 µmol/L), which is critical for reducing the prevalence of sarcopenia and all-cause mortality.^[55]

Lastly, both RT conditions decreased the serum levels of IL-6 and TNF-α. Improvement in inflammation levels was unexpected because pro-inflammatory factors typically rise with increased oxidative stress.^[8] A potential explanation for this effect could be the production of myokines during muscle contraction, which exhibit anti-inflammatory properties.^[56] Nevertheless, IL-6 levels decreased more significantly in the AS group (d = 1.16). The additional reduction in IL-6 may be due to the anti-inflammatory effects of exercise, which are influenced by the redox state. Supplementation with vitamins C and E helped to improve the redox balance, thereby further reducing the levels of inflammatory factors. Alternatively, vitamins C and E may exert direct anti-inflammatory effects.^[52,57] It has been reported that reduced circulating levels of IL-6 play an important role in mitigating muscle mass and strength loss in sarcopenia.^[58]

4.1. Strengths and limitations

High intervention adherence and lack of adverse events were noted during the study, indicating the satisfactory acceptability and safety of elastic band exercise and vitamins C and E supplementation in sarcopenia. However, this study has several limitations. First, the results are specific to older women with sarcopenia and should not be generalized to other populations. Second, our study did not have an antioxidant vitamin supplementation group or an exercise-alone group, and the supplemental doses of vitamins C and E were based on previous studies of healthy older adults. Future research should further explore the synergistic mechanisms of vitamins C and E and RT as well as determine the optimal supplement dosage options for the sarcopenia population. Finally, we did not assess post-test nutritional intake but instructed participants to maintain their regular diet habits throughout the intervention, which was strictly monitored in the training diary.

5. Conclusion

Vitamins C and E supplementation combined with RT may improve muscle mass and strength in older women with sarcopenia, and the underlying mechanism is likely related to the alleviation of oxidative stress and inflammation. Although vitamins C and E have no significant benefits on physical performance, the AS group outperformed the PLA group in all tests. Future studies should investigate if additional improvements in physical performance can be achieved with longer-term interventions. Overall, supplementation with vitamins C and E is valuable in improving RT-induced neuromuscular adaptations and associated risk factors in older women with sarcopenia.

Acknowledgments

The authors wish to thank all the participants in this research project.

Author contributions

Conceptualization: Xu Liu, Bo Chen.

Formal analysis: Yutian Jin, Feiyan Zhong.

Funding acquisition: Xiaohong Chen.

Investigation: Yunjuan Zhang.

Methodology: Yu Li, Yun Zhang, Runhong Cui.

Resources: Bing Wu, Cui Li, Sheng Xu.

Supervision: Xiaohong Chen.

Visualization: Xu Liu, Bo Chen.

Writing – original draft: Xu Liu, Bo Chen.

Writing – review & editing: Xiaohong Chen.