Subjects

In this study, we enrolled elderly women aged sixty to eighty years who exhibited signs of possible sarcopenia. Recruitment took place within local communities from February to June 2023, employing convenient and snowball sampling methods. We utilized advertisements in local community and health service centers, along with telephone invitations for participant recruitment. We screened participants for possible sarcopenia using calf circumference. The target population was identified based on the criteria established by the Asian Working Group for Sarcopenia. Inclusion criteria comprised a calf circumference of < 33 cm or SARC-CalF ≥ 11, along with handgrip strength < 18 kg or/and a 5-time chair stand test ≥ 12 s. Exclusion criteria encompassed individuals who: (1) had recently participated in or were concurrently involved in other clinical trials; (2) had a BMI ≥ 28 kg/m2; (3) suffered from diabetes, severe hypertension, liver decompensation, significantly impaired kidney function (CrCl < 30 mL/min), tumors, cardiopulmonary insufficiency, or gastrointestinal absorption dysfunction; (3) wore a pacemaker, stent, contraceptive ring, or steel nail; (4) had severe lactose intolerance or consumed cheese more than 3 times a week; (5) regularly took vitamin D, calcium tablets, protein powder, fish oil, or other nutritional supplementations; (6) engaged in more than 150 min of moderate-intensity aerobic activity or 75 min of vigorous-intensity aerobic activity per week; (7) were unwilling to adhere to prescribed diets or disagreed with maintaining their activity levels.

Study design

This study was a 90-day, single-site, RCT conducted in the Suzhou Industrial Park community, Jiangsu, China. During enrollment period, face-to-face interview and anthropometric measurements were conducted. Then, participants were randomly assigned to one of three groups: the control group (CG), the original cheese group (OG), and the golden cheese group (GG). The randomization sequence was generated using computer software. The intervention period spanned 90 days, during which measurements were conducted at baseline, midway (60 days), and the end of the trial (Table 1).

Table 1 Study assessment procedures and timetable

All subjects received individual instructions from a dietitian to maintain their habitual diet and refrain from making any major lifestyle changes, especially in terms of exercise. Participants in the OG were instructed to consume 4 slices (66.4 g) of original cheese (Shanghai Milkground Food Tech Co., Ltd, China), containing 9.0 g of protein per 66.4 g. Meanwhile, participants in the GG were directed to consume 4 slices (67.4 g) of golden cheese (Shanghai Milkground Food Tech Co., Ltd, China), which contained 12.7 g of protein per 67.4 g. Cheese was selected because of its convenience, appeal (better tolerated than milk), affordability and acceptability among elderly adults. The chosen amounts of cheese were determined based on assessments for improvements of muscle strength from previous studies [15, 19] and selected to be within a range that participants could feasibly consume. The key nutrients of the intervention cheeses were presented in Table 2. The processing methods for golden cheese and original cheese are the same, but the ingredients and their proportions differe. Specifically, golden cheese contains 60% cheddar cheese, compared to 51% in original cheese, and includes additional calcium-containing phosphates. Thus, in comparison to the original cheese, golden cheese provided higher protein and calcium, and lower carbohydrate and sodium.

Table 2 Nutrient composition (daily intake) of intervention cheese

All participants in the intervention groups received brochures containing cheese recipes and were instructed to ingest 2 slices of intervention cheese at both breakfast and dinner. To enhance compliance, the required cheese and a record sheet (to track cheese intake) were provided every two weeks. Adherence to consumption of at least 80% of the product was considered acceptable. Additionally, participants were instructed to store the cheese in the freezer throughout the trial. Participants in the control group were given an additional requirement to avoid the two intervention cheeses, as well as similar cheeses.

Human ethics and consent to participate declarations

All participants provided their informed consent to participate in the study, which received approval from the Ethics Committee of Soochow University (Approval Code: SUDA20221005H01) and adhered to the ethical standards outlined in the Declaration of Helsinki. The present study was registered in the Chinese Clinical Trial Registry with the registration number ChiCTR2300078720.

Outcome measures

Trained investigators conducted measurements of anthropometric variables following a standardized protocol. The principal investigator and staff involved in the recruitment and follow-up of participants were “blinded”.

Primary outcome

The primary outcome of this study was the change in muscle mass at 60 days and 90 days. (A) Skeletal Muscle Mass Index (SMI): each participant, wearing light clothing and devoid of metal objects, underwent body composition assessment during early morning fasting. Muscle mass was measured using the inBody 770 (Biospace Co., Ltd., Seoul, Korea). SMI was determined on the basis of the Bioelectrical Impedance Analysis (BIA). SMI = appendicular skeletal muscle mass (kg) / height² (m²). (B) Calf Circumference: patients were instructed to assume a standing position, and the maximum diameter of the right calf was measured using a measuring tape.

Secondary outcome

The secondary outcomes were changes of muscle strength, muscle function, and biomakers for sarcopenia.

A)

Maximum handgrip strength.

Measurements were conducted following the manufacturer’s recommendations, utilizing an electronic handgrip dynamometer (Aopi Sporting Goods Co., Ltd., USA).

B)

Physical performance.

Participants’ physical performance was evaluated using the short physical performance battery (SPPB), which consisted of 3 components: (1) a 6-meter usual pace walk. Participants performed the walk component twice, and the average gait speed was calculated. (2) a five-repetition chair stand without using one’s arms, assessing leg-power impairment. Volunteers were instructed to rise from a chair and return to a seated position five times as quickly as possible. Time was measured using a stopwatch with precision to the nearest 0.01 s, and the average of two trials was subsequently calculated. (3) a progressive Test of Standing Balance. Tests of standing balance included tandem, semi-tandem, and side-by-side stands. Categories of all physical performance components were established for each set of measures following the procedures published by Guralnik et al [20].

C)

Biomakers for sarcopenia.

Venous blood samples were obtained at both baseline and the end of the study period. Collection was performed using a 5 mL serum separator tube, and serum was isolated through centrifugation. Insulin-like growth factor 1 (IGF-1) was quantified by Liquid Chromatography Tandem Mass Spectrometry (Waters Xevo TQ-S, Waters Corporation, Ltd., Framingham, USA). C-reactive protein (CRP) and Cystatin C were determined through immune transmission turbidity (Beckman AU5800 Automatic Analyzer, Beckman, Ltd., Fullerton, USA). Interleukin 6 (IL-6) levels were measured using chemiluminescence immunoassay (Hunan Kangqing biological technology Co., Ltd., Liuyang, China). The content of free L-carnitine and acylcarnitine in human serum was assessed by enzyme-linked immunoassay (Shanghai Yansheng Industrial Co., Ltd., Shanghai, China). Procedures and tests were conducted by the same group of nurses and research technicians to minimize methodological variations and ensure consistency across both individual subjects and the entire study cohort.

Blood pressure and biochemical variables

The participants were asked to sit quietly for at least 15 min, and blood pressure (BP) was measured on the right arm three times at 5 min intervals using an automatic BP monitor (Shenzhen Jiakang Technology Co., Ltd., Shenzhen, China). Triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), and creatinine levels were determined using the Hitachi Automatic Analyzer 3100 (Hitachi, Ltd., Tokyo, Japan).

Dietetic, physical activity and comorbidities’ data

Dietary intake was assessed using two-day (one weekday and one weekend day) 24-hour dietary recalls. Experienced dietitians administered the recalls through face-to-face interviews, utilizing valid forms. The macronutrient and mineral (calcium and sodium) content of the diet at baseline, midway (60 days), and the end of the trial were calculated based on the China Food Composition Table (2019) to establish a measure of dietary control, ensuring that habitual nutritional intake did not significantly impact the study findings. To account for any variations in physical activity during the entire trial, a physical activity metabolic equivalent of task (MET) was determined using the International Physical Activity Questionnaire Short Form (IPAQ-SF) at three points (0, 60, 90 days).

Statistical analysis

The sample size of 22 subjects in each group was determined through power analysis, targeting 80% power at a 5% significance level, based on SMI as the desired effect size. Baseline continuous variables were summarized using mean (standard deviation) or median (interquartile range), while categorical parameters were expressed as numbers and proportions. Group differences in participant characteristics were assessed using either one-way ANOVA or the Kruskal–Wallis test.

A general linear model was employed to compare differences in markers of muscle and sarcopenia, serum biomarkers, blood lipid, and glucose among the three groups, adjusting for age, BMI, drinking habits, physical activity, and current main nutrient intake (protein and calcium). Furthermore, between-group comparisons were conducted using either LSD or the Mann-Whitney U test. A general linear model-repeated measures approach was developed to assess differences in self-comparison before and after the intervention, with additional adjustments made for changes in calcium and protein intake, as well as physical activities.

Statistical significance was set at a two-tailed P-value < 0.05. All statistical analyses were performed using SPSS version 26.0 (IBM SPSS, USA).