We compared the effects of one vs two daily bouts of walking on aerobic fitness and body composition in postmenopausal women. One hundred and thirty-four subjects were randomized into exercise groups or a control group and 130 completed the study. The subjects walked 5 d/week for 15 weeks at 65% of their maximal aerobic power expending 300 kcal (1255 kJ) in exercise in one (Group S1) or two daily sessions (Group S2). VO(2max) was measured in a direct maximal treadmill test. Body mass index (BMI) was calculated and the percentage of body fat (fat%) estimated using skinfold measurements. The net change in the VO(2max) was 2.5 mL min/kg (95% CI 1.5, 3.5) (8.7%) in Group S1 and 2.5 mL min/kg (95% CI 1.5, 3.5) (8.8%) in Group S2. The net change in body mass was -1.2 kg (95% CI-1.9, -0.5) in Group S1 and -1.1 kg (95% CI -1.8, -0.4) in Group S2. The net fat% change was -2.1% (95% CI-2.7, -1.4) in Group S1 and -1.7% (95% CI-2.3, -1.0) in Group S2. Exercise improved the maximal aerobic power and body composition equally when walking was performed in one or two daily bouts.