The objective of the study was to examine the association between fatigue (measured by the Multidimensional Fatigue Inventory; MFI-20) and physical activity (measured by the Saltin-Grimby Physical Activity Level Scale; SGPALS) in a large cohort of patients (≥18 years) with myasthenia gravis (MG) including relevant disease - and lifestyle-related factors. A total of 1463 persons, registered at the Danish National Registry of Patients with a MG diagnosis, according to the International Classification of Diseases, received a web-based survey. A total of 779 patients (53% women, mean [SD] age 60.8 [15.5]) responded. The remaining persons were either non-responders (n = 390) or could not confirm the MG diagnosis (n = 294). The most prominent MFI-20 fatigue domains were general fatigue (median [inter-quartile ranges, IQR], 13 [10–16]) and physical fatigue (median [IQR], 13 [9–15]), and 386 (53%) patients reported low levels of physical activity. All fatigue domains were associated with physical activity (p<.01). Higher level of physical activity was associated with lower levels of fatigue. Important factors for the association were myasthenia gravis disease severity (measured by the Myasthenia Gravis Activities of Daily Living profile), body mass index, insomnia (measured by the Insomnia Severity Index) job-status, comorbidity, and cohabitation.