It has been reported that exercise following myocardial infarction (MI) attenuates left ventricular (LV) pump dysfunction by normalization of myofilament function. This benefit could be attributed to an exercise-induced up regulation of endothelial NO synthase (eNOS) expression and activity. Consequently, we first tested the hypothesis that the effects of exercise after MI can be mimicked by elevated eNOS-expression, using transgenic overexpressing mice for human eNOS (eNOSTg). Both exercise and eNOSTg attenuated LV remodeling and dysfunction after MI in mice, and improved cardiomyocyte maximal force development (F_max). However, only exercise training restored myofilament Ca²⁺-sensitivity and SERCA2a protein levels, and improved LVdP/dt at 30 mmHg. Conversely, only eNOSTg improved survival. In view of these partly complementary actions we subsequently tested the hypothesis that combining exercise and eNOSTg would provide additional protection against LV remodeling and dysfunction after MI. Unexpectedly, the combination of exercise and eNOSTg abolished the beneficial effects on LV remodeling and dysfunction of either treatment alone. The latter was likely due to perturbations in the Ca²⁺-homeostasis, as myofilament F_max actually increased despite marked reductions in phosphorylation status of several myofilament proteins, whereas the exercise-induced increases in SERCA2a protein levels were absent in eNOSTg mice. Anti-oxidant treatment with N-acetylcysteine prevented these detrimental effects on LV function, while partly restoring phosphorylation status of myofilament proteins and further enhancing myofilament F_max. In conclusion, the combination of exercise and elevated eNOS-expression abolished the cardioprotective effects of either treatment alone after MI, which appeared to be, at least in part, the result of increased oxidative stress secondary to eNOS 'uncoupling'.