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-h fast on hepatic glucose production (R_a), peripheral glucose uptake (R_d), and skeletal muscle insulin signaling in lean and obese subjects. Healthy lean [n = 14; age = 28.0 ± 1.4 yr; body mass index (BMI) = 22.8 ± 0.42] and nondiabetic obese (n = 11; age = 34.6 ± 2.3 yr; BMI = 36.1 ± 1.5) subjects were studied following a 12- and 48-h fast during 2 h of rest and a 3-h 40 mU·m⁻²·min⁻¹ hyperinsulinemic-euglycemic clamp (HEC). Basal glucose R_a decreased significantly from the 12- to 48-h fast (lean 1.96 ± 0.23 to 1.63 ± 0.15; obese 1.23 ± 0.07 to 1.07 ± 0.07 mg·kg⁻¹·min⁻¹; P = 0.004) and was equally suppressed during the HEC after both fasts. The increase in glucose R_d during the HEC after the 12-h fast was significantly decreased in lean and obese subjects after the 48-h fast (lean 9.03 ± 1.17 to 4.16 ± 0.34, obese 6.10 ± 0.77 to 3.56 ± 0.30 mg·kg FFM⁻¹·min⁻¹; P < 0.001). After the 12- but not the 48-h fast, insulin-stimulated AKT Ser⁴⁷³ phosphorylation was greater in lean than obese subjects. We conclude that 1) 48 h 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.