Background. Children with disabilities have an increased risk for reduced fitness and reduced health-related quality of life (HRQoL). Fitkids, a nationwide exercise therapy program in the Netherlands, was developed to improve fitness and HRQoL in children with disabilities. Objective. The study objective was to determine the effects of the Fitkids program on health-related fitness, walking capacity, and HRQoL in children with disabilities or chronic conditions. Design. This was a quasi-experimental single-group longitudinal study. Methods. Fifty-two children and adolescents who were referred to the Fitkids program participated in this study. Participants received a graded exercise training program for 6 months, with frequencies of 1 hour 2 times per week in the first 3 months and 1 hour per week during months 4 to 6. Health-related fitness (aerobic fitness, anaerobic fitness, and muscle strength), walking capacity, and HRQoL were evaluated at baseline and after 3,and 6 months of training. Multilevel modeling was used to quantify the contributions of repeated measures, participants, and Fitkids centers to variations in health-related fitness, walking capacity, and HRQoL during the intervention period. The models were adjusted for sex, height, and weight. Results. After 6 months of training, significant intervention effects were found for aerobic fitness, anaerobic fitness, and muscle strength. A significant effect also was found for walking capacity. On the HRQoL measure, significant improvements were found for the self-reported and parent-reported physical and emotion domains and for the parent-reported total score for HRQoL. Limitations. No control group was included in this study. Conclusions. The Fitkids exercise therapy program has significantly improved health-related fitness, walking capacity, and HRQoL in children and adolescents with chronic conditions or disabilities.