Aim: Underfeeding is known to reduce resting energy expenditure (REE) as an energy-conserving mechanism and may also reduce insulin sensitivity. Uncoupling protein 1 is known to have a significant role in energy expenditure (EE) in small mammals, but the role of UCPs in humans is unclear. UCP3 is primarily expressed in human skeletal muscle, a significant site of whole-body EE in lean individuals and therefore has a potential role in human metabolism. Here, we examine the effects of short-term underfeeding on UCP3 skeletal muscle expression, and on whole-body insulin sensitivity, substrate utilization and thermogenesis. Methods: Eleven non-obese men [age 22.8 +/- 1.34 years, body mass index 23.4 +/- 0.71 kg/m(2), mean +/- s.e.m.]were fed for two periods of 6 days, an underfeeding diet (UF) (50% predicted requirements for weight maintenance) and an eucaloric diet (EU), with the same macronutrient composition, in random order. Subjects visited the laboratory on four separate occasions, before and after each dietary period. REE, metabolites and muscle biopsies (vastus lateralis) were taken and the thermogenic response to a hyperinsulinaemic euglycaemic clamp was measured over a 2-h period. UCP3 mRNA levels were measured using Taqman. Results: After underfeeding for 6 days, REE fell by 0.43 +/- 0.17 kJ/min (p = 0.032), with weight loss of 2.05 +/- 0.34 kg (p < 0.001). Baseline fasting glucose was significantly lower at 4.26 +/- 0.07 mmol/l (p = 0.005), with a corresponding fall in carbohydrate oxidation (0.08 +/- 0.03 g/min; p = 0.04). Fasting free fatty acids (FFA) increased by 0.13 +/- 0.03 mmol/l ( p < 0.001), with an increase in beta-hydroxybutyrate concentrations of 0.41 +/- 0.07mM(p < 0.001) compared with post-EU. There was no significant change in UCP3 mRNA levels pre- and post-UF [10.4 +/- 6.8 arbitrary units (au); p = 0.16] compared with pre- and post-EU (3.2 +/- 7.3 au; p = 0.67). There was no thermogenic response to the clamp after 6 days of underfeeding and a significant reduction in glucose disposal rates (from 46.35 +/- 2.15 to 39.46 +/- 1.12 mmol/min/kg; p = 0.003). Carbohydrate oxidation rates were lower by 0.08 +/- 0.03 g/min (p = 0.011) compared with pre- UF, with no change in glucose storage rates (28.2 +/- 2.4 mmol/min/ kg pre- UF; 27.0 +/- 2.3 mmol/min/ kg post-UF; p = 0.7). EU resulted in a mildly underfed state with marginal weight loss (0.55 +/- 0.28 kg; p 0.08), and fasting FFA increased by 0.13 +/- 0.03 mmol/l ( p < 0.001) and b-hydroxybutyrate concentrations by 0.05 +/- 0.02mM(p = 0.03) compared with pre- EU. There was no change in glucose disposal or storage rates compared with pre- EU. Conclusions: Underfeeding for 6 days has no significant effect on UCP3 mRNA expression in skeletal muscle in non-obese men but is associated with changes in carbohydrate fuel partitioning, REE and the thermogenic response to the glucose clamp. Mild underfeeding had no effect on insulin sensitivity, but more severe energy restriction reduced insulin-stimulated glucose oxidation without affecting glucose storage.