The myocardial Na⁺/H⁺ exchanger-1 (NHE1) plays a major role in regulation of intracellular pH and its upregulation has been implicated in increased ischemia-reperfusion injury and other pathologies. Hydrogen peroxide (H₂O₂) increases NHE1 activity acutely via ERK1/2 signaling. Chronic strenuous exercise upregulates NHE1 in skeletal muscle, but we hypothesize this will not occur in the heart because exercise creates a cardioprotective phenotype. NHE1 activity and its regulation by H2O2 were examined at physiological pH using isolated cardiomyocytes from female Sprague-Dawley rats exercised on a treadmill for 5 wks (E, n=11). Compared to sedentary (S, n=15), E displayed increases (P<0.05) in heart:body weight (6.8%) and plantaris mitochondria content (89%). NHE1 activity (acid efflux rate following an acid load) was 209% greater in E (0.65±0.12 v 2.01±0.29 fmol/min). The difference was attributed primarily to greater cell volume (22.2±0.6 v 34.3±1.1 pL) and pH_i-buffering capacity (33.94±1.59 v 65.82±5.20 mM/pH unit) of E myocytes. H₂O₂ stimulation (100 µM) raised NHE1 activity significantly less in E (45%) than S (167%); however, activity remained 185% greater in E. ERK1/2 inhibition abrogated the increases. H₂O₂-stimulated ERK1/2 phosphorylation levels normalized to total ERK1/2 were similar between groups. Content of NHE1 and activities of H₂O₂ scavengers were also similar. We observed that pHi-buffering capacity differences between groups became progressively less with declining pH, which may be an exercise-induced cardioprotective adaptation to lower NHE1 activity during certain pathological situations. We conclude that strenuous endurance exercise increases myocardial NHE1 activity at physiological pH, which would likely enhance cardiac performance under physiological conditions.