Effects of an 8-Week Aquatic Training Program and Red Grape Juice Supplementation on Gut Permeability, Inflammatory Markers, and Neuromuscular Performance in Female Patients with Multiple Sclerosis

To investigate the effect of 8 weeks of aquatic exercise and red grape juice supplementation on intestinal permeability, inflammation, and neuromuscular function in women with Multiple Sclerosis.

Randomized, controlled, parallel-group, double-blind clinical trial (blinding of participants to the beverage, and blinding of assessors and analysts), Sample size: 70 (5 groups of 14). Simple randomization using a random number generator software, with allocation concealment via coded bottles.

Exercise in an indoor pool (26-28°C). Blood sampling 48h before/after intervention, after 12h fasting. Measurements: inflammation (IL-2, IL-10, TNF-alpha) and cortisol by ELISA; intestinal permeability (LPS, zonulin) and stool SCFAs by GC; neuromuscular function by EMG-NCV, dynamometer, and balance platform. All measurements by blinded assessors.

Inclusion: Women with RRMS; confirmed 2017 McDonald criteria; EDSS 1-4.5; aquatic exercise capability; no recent relapse; no severe cardiac, metabolic, or GI disease; no antioxidants/regular exercise in 3 months; no grape allergy; signed consent. Exclusion: Relapse during study; medication change; non-cooperation; severe systemic disease; new antioxidant use; exercise outside protocol; smoking.

Five parallel groups (n=14): 1) Aquatic exercise + grape juice, 2) Aquatic exercise + placebo, 3) Supplement only, 4) Placebo only, 5) Control. Exercise: 8 weeks, 3 sessions/week, 60 min at 50-70% HRmax. Supplement: 250 ml grape juice or placebo, 3 times/week.

Intestinal permeability: LPS, zonulin. Systemic inflammation: IL-2, IL-10, TNF-alpha, cortisol. Stool short-chain fatty acids: acetate, propionate, butyrate. Neuromuscular function: nerve conduction velocity (EMG-NCV), muscle strength (dynamometer), balance (Stability Platform).

Simple randomization was performed using a computer-generated random number sequence. Participants were allocated into five groups of 14. For allocation concealment, coded bottles were used, and only one person outside the data collection and analysis process had access to the allocation codes.

In this study, blinding for the nutritional intervention (red grape juice supplement vs. placebo) has been fully implemented. Since there is no 'exercise-alone' group among the exercise groups, and both exercise groups receive a beverage (one supplement and one placebo), participants cannot distinguish whether their beverage is active or not. The supplement and placebo are completely identical in color, taste, volume, and packaging. Furthermore, outcome assessors and data analysts are blinded to the group allocation of the participants. Given the nature of the exercise intervention, blinding to 'performing exercise' is not feasible; however, this limitation has been compensated for by the precise blinding of assessors and analysts.

Public sharing of Individual Participant Data (IPD) is not planned for this study. This decision is made to ensure the confidentiality of participants' personal information and to fully comply with the ethical principles outlined in the informed consent forms. The raw data will be used solely for the statistical analyses of this research and will remain accessible only to the research team.