Mice are among the most widely used translational models of cardiovascular aging and offer a method to quickly assess lifespan changes in a controlled environment. The standard laboratory temperature (20-22 degrees C), however, imposes a cold stress on mice that causes an increase in sympathetic nervous system-mediated activation of brown adipose tissue (BAT) to maintain a core body temperature of 36-37 degrees C. Thus, while physiologic data obtained recapitulate human physiology to a certain degree, interpretations of previous research in mice may have been contaminated by a cold stress, due to housing mice below their thermoneutral zone (30 degrees C). The purpose of this investigation was to examine how chronic sympathetic stimulation evoked by acclimation to 20 degrees C might obscure interpretation of changes in autonomic modulation of heart rate (HR) and heart rate variability (HRV) that accompany advancing age. HR and HRV before and after administration of a dual-autonomic blockade were measured via in-vivo ECG in young (3 months) and aged (30 months) male C57BL/6 telemetry-implanted mice following temperature acclimation for 3 days at 30 degrees C or 20 degrees C. Mean basal and intrinsic HR of both young and aged mice became markedly reduced at 30 degrees C compared to 20 degrees C. In both age groups, HRV parameters in time, frequency, and non-linear domains displayed increased variability at 30 degrees C compared to 20 degrees C under basal conditions. Importantly, age-associated declines in HRV observed at 20 degrees C were ameliorated when mice were studied at their thermoneutral ambient temperature of 30 degrees C. Thus, an accurate understanding of autonomic modulation of cardiovascular functions in mice of advanced age requires that they are housed in a metabolically neutral environment.