Purpose: To verify whether excess body mass influences 24-h ambulatory heart rate variability (HRV) after a moderate-intensity continuous exercise (MICE) session. Method: Participants included 27 non-trained young men that were divided into two groups, 1) normal-weight (n = 10) and 2) overweight and obese (n = 17). Participants underwent a single MICE session of 30 minutes at 50-60% of heart rate reserve (HRR), and a control session (CT). Heart rate (HR) and HRV indices were recorded at 60-minute intervals and were used to obtain the area under the curve (AUC) for 24-h ambulatory measurements following MICE or CT. SDNN (standard deviation of RR intervals in milliseconds) and RMSSD (root mean square of successive differences between adjacent RR interval in milliseconds), Ln-LF (log-transformed values of the absolute power of the low-frequency band) and HF (log-transformed values of the absolute power of the high-frequency band) were analyzed. Results: The overweight and obese group presented higher AUC of RMSSD (p = 0.006), Ln-LF (p = 0.002), and Ln-HF (p = 0.005) indices after MICE than CT. Nighttime periods were more responsive to the effects of MICE on RMSSD and Ln-HF indices (p < 0.05), regardless of group. Conclusion: MICE promoted an increase in the 24-h ambulatory cardiac autonomic modulation in the group overweight and obese, mainly at nighttime during sleep. These results revealed a potential benefit of MICE on the cardiac autonomic modulation for young men with overweight and obesity.