There is growing evidence using human and rodent models that exhaustive exercise can transiently reduce cardiac contractile function, a term aptly named cardiac fatigue. The purpose of this study was to examine the mechanisms associated with exercise-induced cardiac fatigue. Male Sprague-Dawley rats (21-23 weeks) were randomly assigned to a control group (CTL), an acute exercise group (AC) or a group that performed acute exercise and were allowed to recover for 24 hours (24H). The exercise protocol consisted of treadmill running at 5% grade and 20 m/min until exhaustion. A second cohort of animals were assigned to the same experimental groups but were provided 1.5 mM apocynin (APO), a NADPH oxidase (NOX) inhibitor, in their drinking water for 3 days prior to the acute exercise bout. The left ventricle (LV) from anaesthetized rats (35 mg/kg sodium pentobarbital; intravenous) was excised and contractile function was assessed ex vivo using standard isolated perfused heart techniques. Reactive oxygen species (ROS) production, rates of sarco(endo)plasmic reticulum (SR) Ca2+-ATPase activity, -uptake, -release and -leak and calpain activity were assessed in vitro on whole LV homogenate. In the absence of NOX inhibition, cardiac fatigue, as indicated by reductions in left ventricular developed pressure (LVDP) compared with CTL, was observed in AC (-12%) and 24H (-14%) groups. SR Ca2+-ATPase activity, -uptake and -release were not different (p>0.05) between groups; however, SR Ca2+-leak was significantly increased in the AC group and normalized in the 24H group compared to the CTL group (CTL 0.27±0.03; AC 0.38±0.05; 24H 0.23±0.02 µmol/g/min, p<0.05). ROS production (+15%) and calpain activity (+29%) were significantly increased (p<0.05) in the AC group compared to the CTL group and there were no differences (p>0.05) between CTL and 24H groups. NOX inhibition prior to exercise completely prevented cardiac fatigue as LVDP was not different (p>0.05) between any of the APO treated groups. Rates of SR Ca2+-leak, ROS production and calpain activity were also normalized by APO treatment. These data suggest that cardiac fatigue present immediately following exercise is associated with increased SR Ca2+-leak and calpain activation. The cardiac fatigue observed in the 24H group may also be related to the proteolytic activity of calpains. The ability of APO treatment to prevent cardiac fatigue suggests that ROS produced by NOX may have contributed to the cellular and whole heart deficits observed in this study.