Cardiovascular stress in response to treadmill exercise is frequently used to detect cardiac abnormalities that are not readily apparent at rest. Herein we describe a treadmill exercise protocol for mice that allows for quantitation of the performance of an animal and the ability to gather metabolic information in a nonrestraining manner using telemetry implant devices. Transgenic (TG) mice overexpressing ventricular myosin regulatory light chain (MLC2v) were subjected to a 5-wk exercise regimen. The TG mice had significant decreases in their capacity for exercise at relatively high treadmill speeds compared with their nontransgenic (NTG) littermates. There was no indication of a hypertrophic response occurring in TG or NTG animals in response to the exercise protocol, and exercise had no effect on MLC2v phosphorylation. Ultrastructural examination of TG atria showed overtly normal myofibrillar organization but a proliferation of the transverse-axial tubular system. This exercise protocol should prove useful in detecting subtle phenotypes that occur in mice as a result of genetic manipulation of the cardiac compartment.