Rodents are increasingly used in atrial electrophysiology research, yet such studies are often performed under anesthesia owing to technical challenges. Here we developed an implantable device for comprehensive atrial studies in ambulatory rats and investigated the effects of commonly used anesthetics on supraventricular electrophysiology and arrhythmic substrate, comparing them with the unanesthetized state (UAS). Adult rats were evaluated 4 weeks after implantation. Studies were conducted in the UAS under 2% isoflurane (ISO) and under 40 mg/kg pentobarbital (PEN). Pacing protocols determined various parameters, including sinoatrial node recovery time, atrioventricular node effective refractory period and atrial effective refractory period. Arrhythmic substrate was assessed after 20 triggering bursts per condition, and arrhythmic tendency was analyzed manually and through the complexity ratio, an unbiased measure recently developed by our group. PEN mildly increased heart rate in both sexes, while ISO did not affect heart rate but prolonged the corrected sinus node recovery time in males. PEN increased atrioventricular node effective refractory period in both sexes, while ISO affected males only. Both ISO and PEN prolonged atrial effective refractory period compared with UAS in both sexes. Arrhythmic measures were higher in males and were attenuated by ISO and, to a lesser extent, by PEN in males only. The dominant frequency of arrhythmic events was reduced by both anesthetics in both sexes. These findings demonstrate a significant impact of commonly used anesthetics on rat supraventricular electrophysiology, with sex-based differences, highlighting the importance of methodologies that enable cardiac electrophysiology studies in unanesthetized rodents.