Voluntary exercise using running wheels has been widely used to force skeletal muscle adaptation in rodent models. However, running in this training model has been poorly characterized, although it is important in understanding resulting phenotypic adaptations. Aims of our study were to characterize activity patterns in mice with free access to running wheels and phenotypic adaptations of hindlimb muscles. Male C57BL/6 mice were housed individually in cages equipped with running wheels and exposed to 12:12-h photoperiods. Running wheel revolutions were recorded against time on a PC using Labview software. After six weeks, hindlimb muscles were physiologically and biochemically characterized. Mice ran 7.4 km per day in periodic bursts of activity (typically 4 sec on; 2 sec off) during the dark cycle for the duration of the study. This resulted in increased Na+/K+-pump density in tibialis anterior but not gastrocnemius muscle. Soleus and extensor digitorum longus muscles showed improved Ca2+ handling during electrical stimulation. Contracting soleus muscles consumed 1.3-fold more oxygen, yet no significant change in mitochondrial density was found by citrate synthase assay. These results imply that muscle adaptions to voluntary running primarily target cation homeostasis in mice.