OBJECTIVES: To examine heart rate variability (HRV) in unmedicated patients with major depressive disorder (MDD) and its changes after treatment with agomelatine alone and in combination with sedative-hypnotics. METHODS: We recruited 152 physically healthy, unmedicated patients with MDD and 472 age- and sex-matched healthy volunteers. Frequency-domain measures of HRV were obtained during enrolment for all participants and again for MDD patients after 6 weeks of treatment with agomelatine alone and combining sedative-hypnotics. RESULTS: Compared to the controls, unmedicated patients exhibited significantly lower mean R-R intervals, low-frequency (LF) HRV, and high-frequency (HF) HRV, but higher LF/HF ratios. Fifty-six and 49 patients successfully completed agomelatine monotherapy and the combination therapy of agomelatine and sedative-hypnotics, respectively. Between-group analyses showed significant treatment-by-group interactions for LF-HRV, HF-HRV and LF/HF ratio. The results showed a significant increase in HF-HRV after agomelatine monotherapy, a significant decrease in LF-HRV and HF-HRV, and a increase in the LF/HF ratio after combination therapy. CONCLUSIONS: MDD patients had reduced HRV, and the patterns of HRV changes differed between patients treated with agomelatine alone and in combination with sedative-hypnotics. Clinicians should consider HRV effects when adding sedative-hypnotics to agomelatine, which is important for depressed patients who already have decreased cardiac vagal tone.