The Effect of Combined Aerobic–Resistance Training with Leucine-Enriched Whey Protein Supplementation on Muscle-Derived Exosomal microRNAs and Muscle Functional Indices in Older Adults Aged 60–70 Years

To investigate the effect of combined aerobic-resistance training combined with leucine-rich whey protein supplementation on the expression of these microRNAs and muscle function in elderly men aged 60 to 70 years.

The statistical population of the present study includes all elderly men aged 60 to 70 years living in Ahvaz city who have visited the city's sports and rehabilitation centers in the first six months of 1404.

In the present study, the effect of the independent variable (combined aerobic-resistance training with leucine-rich whey protein supplementation) on the dependent variables (levels of muscle-derived exosomal miR-1, miR-133a, and miR-206 and muscle function indices) was investigated.

Inclusion criteria: 1. Age range 60 to 70 years 2. Not participating in regular exercise programs (at least two sessions per week) in the past six months Exclusion criteria: 1. Missing more than two consecutive exercise sessions or more than 20% of the total exercise sessions during the study period 2. Having acute illnesses or exacerbation of chronic illnesses during the study period

1. Exercise group: Combined aerobic-resistance training (no supplementation) 2. Supplementation group: Leucine-rich whey protein supplement (no exercise) 3. Exercise + Supplementation group: Combined aerobic-resistance training with Leucine-rich whey protein supplement 4. Control group: No exercise and no supplementation

Independent variable: • Combined aerobic-resistance training (8 weeks, three sessions per week) • Leucine-rich whey protein supplement (8 weeks, one serving per day) Dependent variables: • Muscle-derived exosomal miR-1 (relative expression) • Muscle-derived exosomal miR-133a (relative expression)

In this study, a quasi-randomized method based on alternating allocation based on the order of referral was used. Participants were randomly assigned to four groups (exercise, supplement, exercise + supplement, and control) in the order of study entry. The randomization unit was individual and stratification was not used due to the homogeneity of the participants. To conceal allocation, the allocation sequence was kept in opaque, numbered envelopes by someone independent of the researcher.

This study was designed as a single-blind study. Given the open-label nature of the exercise interventions and supplement use, blinding of participants and exercise trainers was not possible, and participants were aware of their intervention group. However, the outcome assessors and data analysts were kept completely blind to the group allocation of participants. All anthropometric measurements, muscle performance indices, and Real-Time PCR tests were performed by independent, trained assessors who were unaware of the grouping of participants. Blood samples and performance data were sent to the laboratory and statistical analyst with only specific codes (without mentioning the participant's name or group). The data analyst was also informed of the data grouping after the statistical analyses were completed and the final results were extracted. The principal investigator was unaware of the group allocation of participants until the intervention was completed and data were collected. This method minimised detection and analysis bias in accordance with the CONSORT 2010 statement and IRCT guidelines.

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