Myocardial performance is likely affected by the relative expression of the two myosin heavy chain (MyHC) isoforms, namely α-MyHC and β-MyHC. The relative expression of each isoform is regulated developmentally and in pathophysiological states. Many pathophysiological states are associated with small shifts in the relative expression of each MyHC isoform, yet the functional consequence of these shifts remains unclear. The purpose of this study was to determine the functional effect of a small shift in the relative expression of α-MyHC. To this end, power output was measured in rat cardiac myocyte fragments that expressed ≈12% α-MyHC and in myocyte fragments that expressed ≈0% α-MyHC, as determined in the same cells by SDS-PAGE analysis after mechanical experiments. Myocyte fragments expressing ≈12% α-MyHC developed ≈52% greater peak normalized power output than myocyte fragments expressing ≈0% α-MyHC. These results indicate that small amounts of α-MyHC expression significantly augment myocyte power output.