Running may affect the mood, behavior and neurochemistry of running animals. In the present study, we investigated whether voluntary daily running, sustained over several months, might improve cognition and motor function and modify the brain levels of selected proteins (SOD1, DYRK1A, MAP2, APP and synaptophysin) in Ts65Dn mice, a mouse model for Down syndrome (DS). Ts65Dn and age-matched wild-type mice, all females, had free access to a running wheel either from the time of weaning (post-weaning cohort) or from around 7months of age (adult cohort). Sedentary female mice were housed in similar cages, without running wheels. Behavioral testing and evaluation of motor performance showed that running improved cognitive function and motor skills in Ts65Dn mice. However, while a dramatic improvement in the locomotor functions and learning of motor skills was observed in Ts65Dn mice from both post-weaning and adult cohorts, improved object memory was seen only in Ts65Dn mice that had free access to the wheel from weaning. The total levels of APP and MAP2ab were reduced and the levels of SOD1 were increased in the runners from the post-weaning cohort, while only the levels of MAP2ab and α-cleaved C-terminal fragments of APP were reduced in the adult group in comparison with sedentary trisomic mice. Hence, our study demonstrates that Ts65Dn females benefit from sustained voluntary physical exercise, more prominently if running starts early in life, providing further support to the idea that a properly designed physical exercise program could be a valuable adjuvant to future pharmacotherapy for DS.