We examined the effect of endurance training on energy substrate partitioning during rest and exercise in postmenopausal women. Ten healthy sedentary (55 +/- 1 years old) subjects completed 12 weeks of endurance exercise training on a cycle ergometer (5 d/wk, 1 h/d, 65% peak oxygen consumption [Vo(2)peak]). Whole-body energy substrate oxidation was determined by indirect calorimetry during 90 minutes of rest and 60 minutes of cycle ergometer exercise. Subjects were studied at 65% Vo(2)peak before training and after training at the same absolute exercise intensity (same absolute workload as 65% of pretraining Vo(2)peak) and same relative exercise intensity (65% of posttraining Vo(2)peak). After training, Vo(2)peak increased by 16.3% +/- 3.9% and resting heart rate decreased by 4 beats per minute (P <.05). During exercise at same absolute intensity, mean arterial pressure decreased by 8 mm Hg (P <.05), heart rate decreased by 19 beats per minute (P <.05), energy derived from carbohydrate decreased by 9.6%, and the energy derived from lipid increased by 9.2% (P <.05). Lactate concentration was lower at the same absolute and relative exercise intensities (P <.05). Changes in substrate partitioning during exercise were accomplished without changes in dietary composition, body weight, or body composition. We conclude that endurance training in healthy postmenopausal women who remain in energy balance results in many of the classic cardiopulmonary training effects, decreases the reliance on carbohydrate, and increases lipid oxidation during a given submaximal exercise task without a reduction in body weight.