Obesity in humans is often associated with metabolic inflexibility but the underlying molecular mechanisms remain incompletely understood. The aim of the present study was to investigate how adaptation to prolonged fasting affects energy/nutrient-sensing pathways and metabolic gene expression in skeletal muscle from lean and obese individuals. Twelve lean and 14 non-diabetic obese subjects were fasted for 48 hours. Whole-body glucose/lipid oxidation rates were determined by indirect calorimetry and blood and skeletal muscle biopsies were collected and analyzed. In response to fasting, body weight loss was similar in both groups but the decrease in plasma insulin and leptin, and the concomitant increase in growth hormone were significantly attenuated in obese subjects. The fasting-induced shift from glucose toward lipid oxidation was also severely blunted. At molecular level, the expression of insulin receptor β (IRβ) was lower in skeletal muscle from obese subjects at baseline, whereas the fasting-induced reductions in insulin signaling were similar in both groups. The protein expression of mitochondrial respiratory-chain components, although not modified by fasting, was significantly reduced in obese subjects. Some minor differences in metabolic gene expression were observed at baseline and in response to fasting. Surprisingly, fasting reduced AMP-activated protein kinase (AMPK) activity in lean but not in obese subjects, whereas the expression of AMPK subunits was not affected. We conclude that whole-body metabolic inflexibility in response to prolonged fasting in obese humans is associated with lower skeletal muscle IRβ and mitochondrial respiratory-chain content as well as a blunted decline of AMPK activity.