Objective: Comprehensively and objectively assess physical activity, aerobic fitness and body fat in adolescents and young adults with myelomeningocele and to investigate their relationships. Design: Cross-sectional study. Subjects: Fifty-one persons (26 males) with myelomeningocele aged 21.1 (standard deviation) 4.5) years. Methods: Physical activity was measured with an accelerometry-based activity monitor. Aerobic fitness was defined as the maximum oxygen uptake during the last minute of a maximal exercise test. Body fat was assessed using sum of 4 skin-folds and body mass index. Correlations were studied using multiple regression analyses. Results: Thirty-nine percent of the participants were inactive and another 37% were extremely inactive. Aerobic fitness was 42% lower than normative values and 35% were obese. Ambulatory status was related to daily physical activity (beta = 0.541), aerobic fitness (beta = 0.397) and body fat (beta = -0.243). Gender was related to aerobic fitness (beta = -0.529) and body fat (beta = 0.610). Physical activity was related to aerobic fitness in non-ambulatory persons with myelomeningocele (beta = 0.398), but not in ambulatory persons. Conclusion: Adolescents and young adults with myelomeningocele were physically inactive, had poor aerobic fitness and high body fat. Differences exist between subgroups regarding gender and ambulatory status.