The positive effects of chronic endurance exercise training on health and performance have been well documented. However, these positive effects have been evaluated primarily at the structural level, and it remains poorly understood how the heart muscle adapts mechanically to exercise training. In order to gain some understanding, we subjected three-month-old Sprague-Dawley rats to treadmill running for eleven weeks at one of three exercise volumes (moderate, high, and overtraining). Following training, hearts were excised and mechanical testing was completed on skinned trabecular bundles. Animals in the overtraining group experienced a significant loss in body mass, a withdrawal from food and drink, and became less active. The control, moderate, and high duration groups responded with a dose-dependent increase in heart mass and passive stresses, with no difference in active stress production. These trends were all reversed in the overtrained animals, despite presenting the greatest fitness on a graded treadmill test.