In humans, -adrenergic stimulation increases energy and fat metabolism. In case of ₁-stimulation, this is fuelled by an increased lipolysis. We examined the effect of ₂-adrenergic stimulation, with and without a blocker of lipolysis, on thermogenesis and substrate oxidation. Furthermore, the effect of ₁- and ₂-adrenergic stimulation on UCP3 mRNA expression was studied. Nine lean males received a 3h-infusion with dobutamine (DOB, ₁) or salbutamol (SAL, ₂). Also, we combined SAL with acipimox to block lipolysis (SAL+ACI). Energy and substrate metabolism were measured continuously, blood was sampled every 30 minutes and muscle biopsies were taken before and after infusion. Energy expenditure significantly increased by ~13% in all conditions. Fat oxidation increased by 47 ± 7% in the DOB group and by 19 ± 7% in the SAL group but remained unchanged in the SAL+ACI condition. Glucose oxidation decreased by 40 ± 9% upon DOB, remained unchanged during SAL and increased by 27 ± 11% upon SAL+ACI. Plasma FFA levels were increased by SAL (57 ± 11%) and DOB (47 ± 16%), whereas SAL+ACI caused ~4-fold lower FFA levels compared to basal levels. No change in UCP3 was found after DOB or SAL, while SAL+ACI down-regulated skeletal muscle UCP3 mRNA levels by 38 ± 13%. In conclusion, ₂-adrenergic stimulation directly increased energy expenditure, independent from plasma FFA levels. Furthermore, this is the first study demonstrating a down-regulation of skeletal muscle UCP3 mRNA expression after lowering plasma FFA concentrations in humans, despite an increase in energy expenditure upon ₂-adrenergic stimulation.