The development of insulin resistance in the obese individual could impair the ability to appropriately adjust metabolism to perturbations in energy balance. We investigated a 12 vs. 48 hr fast on hepatic glucose production (Ra), peripheral glucose uptake (Rd), and skeletal muscle insulin signaling in lean and obese subjects. Healthy lean (n = 14; age = 28.0+/-1.4 yr; BMI = 22.8 +/-0.42) and non-diabetic obese (n = 11; age = 34.6+/-2.3 yr; BMI = 36.1 +/-1.5) subjects were studied following a 12 and 48 hr fast during 2 hrs of rest, and a 3 hr 40mU/m2/min hyperinsulinemic-euglycemic clamp (HEC). Basal glucose Ra decreased significantly from the 12 to 48 hour fast (lean 1.96 +/- 0.23 to 1.63 +/- 0.15 mg/kg/min; obese 1.23 +/- 0.07 to 1.07 +/- 0.07 mg/kg/min, p=0.004) and was equally suppressed during the HEC after both fasts. The increase in glucose Rd during the HEC after the 12 hour fast was significantly decreased in lean and obese subjects after the 48 hour fast (lean 9.03 +/- 1.17 to 4.16 +/- 0.34, obese 6.10 +/- 0.77 to 3.56 +/- 0.30 mg/kg FFM/min, p<0.001). After the 12, but not the 48hr fast, insulin stimulated AKTser473 phosphorylation was greater in lean than obese. We conclude: 1) 48 hours of fasting produces a marked decline in peripheral insulin action, while suppression of hepatic glucose production is maintained in lean and obese men and women, and 2) the magnitude of this decline is greater in lean vs. obese subjects.