The aim of this study was to evaluate in premenopausal women (10 sedentary obese women) the effects of 10 days of exercise on the suppression of whole body and regional lipolysis by insulin. Lipolysis was determined using 2H5-glycerol infusion and microdialysis of sc adipose tissue during a two-stage hyperinsulinemic-euglycemic clamp [10 (LO) and 20 (MO) mU/m·min]. Microdialysis probes were positioned in abdominal and femoral sc adipose tissue to monitor interstitial glycerol and blood flow. Basal plasma glycerol was 86.7 ± 17.0 and 100.3 ± 19.8 μmol/L before and after training, respectively (P < 0.05). Plasma glycerol was suppressed to a greater extent after [to 47 ± 5% (LO) and 42 ± 5% (MO) of basal] than before [to 62 ± 8% (LO) and 55 ± 8% (MO) of basal] training. The rate of appearance of glycerol was suppressed to 49 ± 7% and 40 ± 5% of basal during LO and to 38 ± 5% and 30 ± 4% of basal during MO (P < 0.05) before and after training, respectively. There were no differences in the suppression of lipolysis in abdominal as well as femoral sc adipose tissue as evidenced by similar reductions in dialysate glycerol levels before and after training in each of these tissues. The results indicate that the antilipolytic response to insulin can be improved through endurance exercise training. Intraabdominal adipose tissue or skeletal muscle may be the site of improved antilipolytic response to insulin after training, as improvement was not evident in abdominal or femoral sc adipose tissue.