Neuromuscular and Multi-Omics Synergy of Combined Creatine × HMB Supplementation Plus Exercise to Improve Muscle Function in Sarcopenic Frailty

The SPPB assesses lower-extremity physical function through balance, gait speed, and chair stand components, generating a total score ranging from 0 to 12 (higher scores indicate better physical performance). Only the total score will be analyzed as the primary outcome.

Time in seconds to rise from a chair, walk 3 meters, turn around, return, and sit down. Lower times indicate better mobility and balance.

Appendicular lean mass (sum of arms and legs lean tissue) measured in kilograms using DEXA. Higher values indicate greater muscle mass in limbs.

Maximal isometric handgrip strength measured in kilograms using a validated hydraulic dynamometer (e.g., Jamar Plus+). Each test performed with the dominant hand in seated position, elbow at 90°. Best of three attempts recorded.

Maximal isometric strength of leg and back extensors assessed in kilograms using a back/leg dynamometer (e.g., Baseline Evaluation Dynamometer). Participants stand on the platform and pull a fixed bar upwards. Best of two attempts recorded.

Dynamic strength/endurance of upper limbs measured as the number of full arm curl repetitions in 30 seconds using a 2 kg dumbbell for women and a 4 kg dumbbell for men. Participants are seated upright without armrests.

Measure of independence in activities of daily living (feeding, bathing, mobility, toileting, etc.). Score ranges from 0 (total dependence) to 100 (independent).

Validated tool to screen and assess nutritional status in older adults. Score ranges from 0-30 (higher = better nutritional status).

Cognitive function assessed using the Mini-Mental State Examination (MMSE). Scores range from 0 to 30, with higher scores indicating better cognition.

26-item validated questionnaire covering physical, psychological, social and environmental domains.

Scores range from 26 to 130; higher scores indicate better perceived quality of life.

Whole-body lean soft tissue mass measured in kilograms using DEXA (Hologic Discovery or GE Lunar system) with manufacturer's software and standardized positioning. Higher values indicate greater muscle mass.

Whole-body fat mass measured with DEXA and expressed as a percentage of total body weight. Higher values indicate greater adiposity.

Visceral adipose tissue (VAT) volume estimated by DEXA in the android region of interest, expressed in cubic centimeters (cm³). Higher values indicate greater central adiposity.

Whole-body BMD measured by DEXA, expressed as grams per square centimeter (g/cm²). Higher values indicate greater bone mineral density.

Cognitive function assessed using the MEC (Spanish version of the MMSE). Scores range from 0 to 35, with higher scores indicating better cognition.

Incidence of falls recorded by nursing staff during the study period. Unit: number of events per participant. Lower numbers indicate better outcomes.

Incidence of hospital admissions for any cause. Unit: number of events per participant. Lower numbers indicate better outcomes.

Number of documented respiratory and/or urinary tract infections. Unit: number of events per participant. Lower numbers indicate better outcomes.

Number of new pressure ulcers recorded by nursing staff. Lower numbers indicate better outcomes.

Occurrence of urinary and/or fecal incontinence episodes recorded in clinical charts. Lower numbers indicate better outcomes.

CRP will be measured in serum samples using a high-sensitivity ELISA. Results expressed in mg/L. Higher values indicate greater systemic inflammation.

Serum IL-6 concentration will be measured by ELISA and expressed in pg/mL. Higher values indicate greater systemic inflammation.

Serum TNF-α concentration will be determined by ELISA and expressed in pg/mL. Higher values indicate greater systemic inflammation.

Plasma reduced glutathione measured by colorimetric assay and expressed in µmol/L. Higher values indicate better antioxidant status.

Plasma oxidized glutathione measured by colorimetric assay and expressed in µmol/L. Higher values indicate greater oxidative stress.

Lipid peroxidation index measured as TBARS and expressed in nmol MDA equivalents/mL of plasma. Higher values indicate greater oxidative stress.

Plasma MDA determined spectrophotometrically and expressed in µmol/L. Higher values indicate greater oxidative stress.

Plasma TAC measured using colorimetric assay and expressed as mmol Trolox equivalents/L. Higher values indicate better antioxidant defense.

Changes in the relative expression levels of selected circulating microRNAs associated with muscle metabolism, neuromuscular function, inflammation, oxidative stress, and anabolic signaling pathways. MicroRNAs will be quantified from peripheral blood samples using molecular profiling techniques.

Changes in the relative expression levels of selected exosome-derived microRNAs related to muscle function, inflammation, and metabolic regulation, analyzed from circulating extracellular vesicles isolated from plasma samples.

Analysis of the frequency of selected genetic variants related to muscle metabolism, anabolic signaling, and exercise responsiveness to explore interindividual variability in functional and molecular responses to creatine and HMB supplementation.

Changes in the relative abundance of selected circulating proteins involved in muscle metabolism, inflammation, oxidative stress, and functional adaptation in response to creatine and HMB supplementation combined with exercise. Protein abundance will be assessed using targeted proteomic approaches.