© 2021 IACFS/ME.Objectives: Fatigue and physical behavior are essential factors affecting disability levels in people with multiple sclerosis. The relationship between fatigue and physical behavior has not been investigated systematically. The aim of the study was to determine the associations between fatigue severity and physical behavior in MS and compare fatigue and physical behavior of MS patients with healthy controls. Method: Forty individuals with MS with an Expanded Disability Status Scale ≤2 and 30 healthy controls were included in this cross-sectional study. Fatigue was evaluated with the Fatigue Severity Scale (FSS). The effects of fatigue on daily function were assessed by the Fatigue Impact Scale (FIS). Physical behavior was measured with an accelerometer, and outcomes were categorized by the amount of activity, activity category, and energy consumption. Results: MS subjects reported more fatigue as compared to healthy controls on the FSS (p = 0.000), and fatigue affected daily function more in MS as compared to controls (FIS: p = 0.000). MS participants showed lower scores on all dimensions of physical behavior (p < 0.05). Fatigue was associated with functional limitations (p < 0.05), objective physical activity level, sedentary and moderate activities (p < 0.05), and energy consumption (p < 0.05) in MS. Conclusions: Individuals with MS with no or minimal disability reported more fatigue and less active physical behaviors than healthy controls. Fatigue was inversely associated with physical activity level, moderate activity and, energy consumption. Fatigue was positively associated with sedentary activity and functional limitations in MS. Clinicians should be aware of these relationships when planning rehabilitation strategies. Trial registration:ClinicalTrials.gov identifier: NCT03255460.