Like many biological processes, heart rate exhibits a significant cyclical variation over a 24 h period (circadian rhythm). The mechanism of this tight regulation is debated, with the prevailing opinion suggesting that it is controlled by autonomic neural signalling from the brain. However, an alternate mechanism of altered ion channel signalling within the heart’s own pacemaker cells has been proposed. Currently, no data exists to directly address whether a circadian rhythm exists in autonomic nervous signalling, preventing any conclusive determination of the mechanism behind heart rate variations. However, we have recently established protocols to undertake specific, direct and concurrent recordings of cardiac sympathetic and vagal parasympathetic nerve activity. The right cardiac sympathetic nerve can be isolated via a thoracotomy, and the right vagal nerve similarly exposed via a neck incision, in order to place platinum bipolar recording electrodes to directly record autonomic nerve activity. Using male Sprague-Dawley rats (350-450g) nerve activity was assessed at Zeitgeber time (ZT) 3 and ZT15, when previous experiments have shown heart rate differences peak. ZT0 indicates lights-on and ZT12 indicates lights off under a 12 h:12 h light:dark lighting regime. Measures were undertaken under sodium pentobarbital anaesthesia (80 mg/kg) delivered intraperitoneally, maintained with a variable pentobarbital infusion at ~0.6 mg/kg/min via the jugular vein, and rats were temperature controlled and mechanically ventilated. Emerging data suggests that while a subtle shift in the balance between sympathetic and parasympathetic inputs to the heart may contribute to circadian heart rate regulation, autonomic changes are unlikely to account for the significant heart rate cycle. Neither cardiac sympathetic (ZT3: 35.3 ± 10.1 vs. ZT15: 52.3 ±14.0 Hz, mean ± SEM, n=6, unpaired t-test NS) nor vagal parasympathetic (ZT3: 30.7 ± 10.9 vs ZT15: 26.0 ± 10.1 Hz, mean ± SEM, n=8, unpaired t-test NS) nerve firing rate suggest a primary role for autonomic modulation in circadian heart rate regulation. Currently, the underlying mechanisms of the circadian rhythm in heart rate are debated due a lack of data to conclusively delineate the importance of neural and pacemaker processes. Direct autonomic nerve recordings provide crucial insight into the fundamental mechanisms of heart rate regulation.