Objective: Regular exercise enhances cardiac function and modulates myocyte growth in healthy individuals. The purpose of the present study was to assess contractile function and expression of selected genes associated with intracellular Ca²⁺ regulation after intensity controlled aerobic endurance training in the rat. Methods: Female Sprague–Dawley rats were randomly assigned to sedentary control (SED) or treadmill running (TR) 2 h per day, 5 days per week for 2, 4 or 13 weeks. Rats ran 8-min intervals at 85–90% of VO_2max separated by 2 min at 50–60%. Myocyte length, intracellular Ca²⁺ (Fura-2), and intracellular pH (BCECF) were measured in dissociated cells in response to electrical stimulation at a range of stimulation rates. Results: The increase in VO_2max plateaued after 6–8 weeks, 60% above SED. After 13 weeks, left and right ventricular weights were 39 and 36% higher than in SED. Left ventricular myocytes were 13% longer, whereas width remained unchanged. After 4 weeks training, myocyte contractility was approximately 20% higher in TR. Peak systolic intracellular Ca²⁺ and time for the decay from systole were 20–35 and 12–17% lower, respectively. These results suggest that increased myofilament Ca²⁺ sensitivity is the dominant effect responsible for enhanced myocyte contractility in TR. Intracellular pH progressively decreased as stimulation frequency was increased in the SED group. This decrease was markedly attenuated in TR and the intracellular pH was significantly higher in the TR group at a stimulation rate of 5–10 Hz. This effect may contribute to the increased contractility observed at the higher stimulation frequencies in TR. A higher intrinsic myofilament Ca²⁺ sensitivity was observed in permeabilised myocytes from the TR group under conditions of constant pH and [Ca²⁺]. Western blot analysis indicated 21 and 46% higher myocardial SERCA-2 and phospholamban, but unaltered Na⁺/Ca²⁺-exchanger levels. Competitive RT-PCR revealed that TR significantly increased Na⁺/H⁺-exchanger mRNA. Conclusion: Intensity controlled interval training increases cardiomyocyte contractility. Higher myofilament Ca²⁺-sensitivity, and enhanced Ca²⁺-handling and pH-regulation are putative mechanisms. Our results suggest that physical exercise induces adaptive hypertrophy in cardiac myocytes with improved contractile function.