Physical activity is frequently used as a strategy to decrease pathogenesis and improve outcomes in chronic pathologies such as metabolic or cardiac diseases. In mice, it has been shown that voluntary wheel running (VWR) could induce an aerobic training effect and may provide a means of exploring the relationship between physical activity and the progression of pathology, or the effect of a drug on locomotor activity. To the best of our knowledge, in vivo magnetic resonance imaging (MRI) and other non-invasive methods had not been investigated for training evaluation in mice; therefore, it was proposed to test an MRI method coupled with a cardiorespiratory gating system on C57BI/6 mice for in vivo heart anatomical and functional characterization in both trained and untrained animals. Twenty mice were either assigned to a 12-week VWR program or to a control group (CON - no wheel in the cage). At week 12, MRI scans showed an increase in the left ventricular (LV) wall mass in the VWR group compared with the CON group. The ex vivo measurements also found an increase in the heart and LV weight, as well as an increase in oxidative enzyme activities (i.e. cytochrome c oxidase [COx] in the soleus). In addition, correlations have been observed between ex vivo LV/body weight ratio, COx activity in the soleus and in vivo MRI LV wall mass/body weight. In conclusion, mouse cardiac MRI methods coupled with a cardiorespiratory gating system are sufficiently effective and feasible for non-invasive, training-induced heart hypertrophy characterization, and may be used for longitudinal training level follow-up in mouse models of cardiovascular and metabolic diseases.