Changes in heart rate variability (HRV) indicative of altered autonomic tone are seen chronic obesity, but changes in HRV due to acute overeating are less certain. This study determined if acute overeating of a high-fat diet (HFD) altered HRV during the day and/or night. Adult female New Zealand white rabbits (n=7) consumed a control diet (CONT) before switching to an ad-lib HFD. Using telemetry, 3-min ECG segments were recorded every 20 min for 3 d of CONT and the first 3 d of HFD. Data were analyzed during the day and night for heart rate (HR), RR interval standard deviation (RRISD), RMS of successive RR interval differences (RMSSD), low frequency (LF) and high frequency power (HF), and LF/HF. HFD and respective CONT periods were compared by paired t-test. (*p≤0.05) Table 1. HRV was altered by HFD more at night than during the day. At night, HR increased 37% and RRISD, RMSSD, and HF decreased 60%, 66%, and 86%, respectively. LF/HF increased 170%. HFD-induced daytime changes showed similar trends, but differences were not significant. Therefore, acute overeating of a HFD produced immediate nighttime HRV changes indicating parasympathetic withdrawal and a shift toward sympathetic dominance. Funded by NIH HL64913.