Whereas novel pathways of pathological heart enlargement have been unveiled by thoracic aorta constriction in genetically modified mice, the molecular mechanisms of adaptive cardiac hypertrophy remain virtually unexplored and call for an effective and well-characterized model of physiological mechanical loading. Experimental procedures of maximal oxygen consumption (V˙o _2 max) and intensity-controlled treadmill running were established in 40 female and 36 male C57BL/6J mice. An inclination-dependent V˙o _2 maxwith 0.98 test-retest correlation was found at 25° treadmill grade. Running for 2 h/day, 5 days/wk, in intervals of 8 min at 85–90% of V˙o _2 max and 2 min at 50% (adjusted to weekly V˙o _2 max testing) increasedV˙o _2 max to a plateau 49% above sedentary females and 29% in males. Running economy improved in both sexes, and echocardiography indicated significantly increased left ventricle posterior wall thickness. Ventricular weights increased by 19–29 and 12–17% in females and males, respectively, whereas cardiomyocyte dimensions increased by 20–32, and 17–23% in females and males, respectively; skeletal muscle mass increased by 12–18%. Thus the model mimics human responses to exercise and can be used in future studies of molecular mechanisms underlying these adaptations.