Background: In most cases, multiple sclerosis (MS) patients reduce physical activity with disease progression and many patients are found to be vitamin D deficient. The aim of this study was to evaluate the effect of daily physical activity in everyday life on 25-hydroxyvitamin-D3 (25(OH)D3) serum levels in mildly disabled patients with an Expanded Disability Status Scale (EDSS) ≤ 4.Methods: We analyzed serum 25(OH)D3 levels and recorded daily physical activity (activity duration, number of steps, distance, total calorie consumption) using an activity tracker for 14-days in 25 women and 15 men. Participants recorded their daily sun exposure time by diary during the study period.Results: We found a positive correlation between physical activity and 25(OH)D3 levels in both, Pearson correlation (r = 0.221) and multivariate regression analysis (B = 0.308, β = 0.236), which was stronger than correlation with sunlight exposure (B = -0.044, β = -0.081). EDSS and physical activity were weakly correlated (r = -0.228), but no correlation between EDSS and 25(OH)D3 levels was found (r = -0.077). There were no relevant differences in physical activity (p = 0.803) and 25(OH)D3 concentrations (p = 0.385) between the EDSS groups 0 – 1.5 and 2.0 – 4.0.Conclusion: Physical activity has an independent effect on vitamin D levels in MS patients with low-grade disability.