Studies conducted on anaesthetized animals have shown that neurons within the rostral ventromedial medulla (RVMM) can modulate the cardiac sympathetic outflow. Disinhibition of RVMM neurons by microinjection of the GABA-A antagonist bicuculline methiodide induces a strong increase in heart rate (HR), but inhibition of the same neurons by microinjection of the GABA-A agonist muscimol does not produce clear effects on HR. However, general anaesthesia affects sympathetic outflow and does not permit a evaluation of heart rate variability (HRV) measure. In the present study the role of RVMM neurons in mediating tonic cardiac sympathetic outflow has been assessed in free behaving rats. HRV has been evaluated in six free behaving rats before and after microinjection of muscimol (1mM) within the RVMM. HRV was analyzed within both the time domain (R-R mean; R-R standard deviation (STD-RR); root mean square successive difference (RMSSD)) and frequency domain (High (HF, 0.6–2.4 Hz) and Low Frequency (LF, 0.06–0.6 Hz) bands). Effects of injections were separated into six temporal blocks according to the dynamics of changes in core temperature following RVMM inhibition. At the end of the experiment, animals the site of injection was histologically verified. The power of the LF band, expression of cardiovascular sympathetic drive, was clearly reduced following RVMM neurons inhibition, suggesting that RVMM neurons participate in maintaining tonic cardiac sympathetic outflow.