Exercise evokes adaptations intrinsic to the myocardium that protect against ventricular arrhythmia, yet the underlying mechanisms are not completely understood. We have previously shown that the transition to arrhythmia occurs concomitant with a collapse in mitochondrial membrane potential (ΔΨm). As our previous studies indicated that exercise preserves intracellular redox homeostasis, which directly influences mitochondrial energetics, we hypothesized that rats exposed to exercise (Ex, 10 d of treadmill running) would be protected against reperfusion arrhythmia via better maintenance of ΔΨm. To fully understand the temporal relationship between ΔΨm and cardiac electrical activity, two-photon microscopy images (using the fluorescent probe TMRM) and volume-conducted electrocardiogram were simultaneously recorded. Langendorff-perfused hearts underwent 40/30 min of ischemia/reperfusion. Exercise lowered the incidence of arrhythmia, with 3 of 8 Ex hearts experiencing tachycardia or fibrillation compared to 7 of 8 sedentary (Sed) hearts. Ex prevented the collapse of ΔΨm during the first 10 min of reperfusion (74±6.4% v 57±1.5% of baseline fluorescence intensity; P<0.05). To gain a more comprehensive understanding of energetics throughout the heterogeneous mitochondrial population, we then measured mean TMRM fluorescence intensity in isolated ventricular mitochondria harvested after reperfusion using flow cytometry (n=100,000 events per group). Interestingly, mean fluorescence intensity for ΔΨm was similar in Ex and Sed mitochondria (278±33 v 309 ±44 AU, respectively). Mitochondrial respiratory control ratios were also similar in Ex and Sed (9.03±0.70 v 9.00±0.92, respectively). Taken together, the isolated mitochondrial assessment did not reflect what was observed in vivo. This suggests that either intracellular factors influenced in vivo mitochondrial energetics, or our isolated mitochondria may have been enriched with predominantly healthy mitochondria. Our studies demonstrate for the first time that exercise prevents electrical dysfunction following an ischemic insult through better preservation of mitochondrial energetics, and that this preservation is only observed in the intact organ.