Stimulation of beta-adrenergic receptors (-AR) by the sympathetic nervous system (SNS) modulates energy expenditure (EE), but substantial inter-individual variability is observed. We determined if the thermogenic response to -AR stimulation is related to genetic variation in codon 16 of the ₂-AR, a biologically important -AR polymorphism, and if differences in SNS activity (i.e., the stimulus for agonist-promoted downregulation) are involved. The increase in EE (EE - indirect calorimetry, ventilated hood) above resting EE in response to non-specific -AR stimulation (intravenous isoproterenol: 6, 12 and 24 ng/kg fat-free mass (FFM)/min) was measured in 46 healthy adult humans (Arg16Arg: 9 male, 7 female, 48±5 years; Arg16Gly: 11 male, 4 female, 53±5 years; Gly16Gly: 3 male, 12 female, 48±5 years (mean±SE)). Neither FFM-adjusted baseline resting EE (P=0.83) nor the dose of isoproterenol required to increase EE 10% above resting (P=0.87) differed between the 3 groups (Arg16Arg: 5409±209 kJ/day, 11.2±2.1 ng/kg FFM/min; Arg16Gly: 5367±272 kJ/day, 11.1±2.1 ng/kg FFM/min; Gly16Gly: 5305±159 kJ/day, 10.5±2.1 ng/kg FFM/min). Consistent with this, muscle sympathetic nerve activity and plasma norepinephrine concentrations were not different among the groups. Group differences in sex composition did not influence the results. Our findings indicate that the thermogenic response to non-specific -AR stimulation, an important mechanistic component of overall -AR modulation of EE, is not related to this ₂-AR polymorphism in healthy humans. This may be explained in part by a lack of association between this gene variant and tonic SNS activity.