Youth with chronic diseases or physical disabilities (CDPD) often show reduced fitness and physical activity (PA) levels and participate less in organized sports compared with healthy peers. The purpose of this study was to examine the associations between participation in sports and health-related fitness and PA in youth with CDPD. A total of 163 subjects (mean age 14 years; range 8-19 years) with CDPD were included in this cross-sectional study, with 81 participating in organized sports and 82 not. Subjects were recruited between October 2014 and November 2016. Aerobic and anaerobic fitness, agility, and muscle strength were assessed in the laboratory, whereas PA was monitored in daily life using accelerometry during 1 week. Linear regression analyses were used to assess the associations of sports participation (independent variable) with health-related fitness and PA (dependent variables). Results show that youth with CDPD participating in organized sports 2 times a week performed better on all outcome measures. They reached a higher peak oxygen uptake (difference of 4.9 ml O2·kg-1·min-1, P = 0.001) compared with their peers not participating in sports. Also, anaerobic fitness, agility, muscle strength, and PA were all positively associated with sports participation. Moreover, the association between sports participation and aerobic fitness was mediated by PA for 31% (P = 0.045). In conclusion, participation in sports is associated with both higher levels of PA and health-related fitness in youth with CDPD. Promotion and stimulation of participation in sports seems a good way to promote health-related fitness as well as a healthy active lifestyle in youth with CDPD.