Background: Moderate and vigorous physical activity is associated with improved outcomes in youth with multiple sclerosis (MS). Physical fitness may also influence disease and health outcomes in this population. Objectives: To determine if there were differences in physical fitness between youth with MS and healthy controls (HC). To examine relationships between physical fitness, physical activity (PA) level, fatigue, depression and disease activity in youth with MS and HC. Methods: Youth with MS ( n = 19) and HC ( n = 21) completed tests establishing cardiorespiratory-fitness (VO2peak), endurance via 2-minute walk test, and musculoskeletal strength via grip strength (GS). Questionnaires determined fatigue, depression, and PA levels. Weekly PA level was determined by accelerometry. Tests of differences and correlational analyses were used to evaluate physical fitness. Results: Youth with MS had lower VO2peak ( U = 279, p < 0.0001), endurance ( t = 2.6, p = 0.02), and higher body mass index (BMI) ( t = -5.9, p = 0.001) than HC. Higher VO2peak was associated with higher moderate to vigorous PAaccelerometer in HC (Spearman-Rho = 0.5, p = 0.03), but not in youth with MS (Spearman-Rho = 0.5, p = 0.06). Lower VO2peak and GS were associated with higher disability (Spearman-Rho = -0.6, p = 0.03) and relapses in MS (Spearman-Rho = -0.52, p = 0.04). Conclusions: Youth with MS have lower levels of fitness, compared with HC. Higher levels of fitness were associated with lower disease activity and disability in youth with MS.