Objective: To explore associations between physical activity and peak oxygen uptake (VO2peak), age, sex, and Hoffer classification in young wheelchair-users with spina bifida. Design: Exploratory study. Subjects: Fifty-three dutch children (age 5-19 years) with spina bifida who use a manual wheelchair. Methods: For the dependent variable physical activity, data from 2 physical activity monitors were analysed: VitaMove data for 34 participants and Actiheart data for 36 participants. Time sedentary, time physically active, and time in moderate to vigorous physical activity were analysed. The Wheelchair Shuttle Test was used to measure VO2peak. Univariate and multivariate regression analyses were performed. Independent variables were VO2peak, age, sex, and Hoffer classification. Results: Time sedentary and time physically active during a school day were influenced by age (β=0.326/β=-0.320) and Hoffer classification (β=0.409/β=-0.534) and during a weekend day by Hoffer classification (β=0.617/β=-0.428). Time in moderate to vigorous physical activity was influenced by Hoffer classification (β=-0.527) during a school day and by age (β=-0.600) during a weekend day. Conclusion: Older age and the inability to walk negatively influence physical activity. Sex and VO2peak were not associated with physical activity. These results imply that increasing cardiorespiratory fitness alone will not improve physical activity in young wheelchair-users with spina bifida.