Background: The autonomic nervous system plays a central role in the maintenance of hemodynamic stability. Cardiac autonomic dysfunction may result in serious complications, such as sudden cardiac death. Heart rate variability (HRV) is significantly reduced in patients undergoing chronic hemodialysis (HD). The aim of this study was to evaluate the effect of on-line hemodiafiltration (OL-HDF) on the autonomic nervous system in chronic HD patients. Methods: Forty chronic HD patients were prospectively studied. The participants were divided into conventional HD and OL-HDF groups. They received regular high-flux HD or OL-HDF for 4-hour sessions, three times a week. Time-and frequency-domain measures of the 24-hour HRV were analyzed during the interdialytic period prior to postdilution OL-HDF and every 6 months for 24 months. The 7-year survival was also evaluated. Results: Among the 40 participants, 15 patients in the HD group and 11 patients in the OL-HDF group completed the study. There was no difference in the baseline characteristics. After 24 months of treatment, β2-microglobulin concentration decreased (from 33.4715.2 mg/dL to 28.476.2mg/dL, P ¼0.02) in the OL-HDF group, while there was no change in the HD group. In the HRV analysis, the frequency-domain HRV parameters increased significantly compared with baseline in the OL-HDF group [natural logarithmic high frequency (lnHF), 3.1573.36 ms2 vs. 4.4273.81 ms2; ln low frequency (LF), 3.567 3.17 ms2 vs. 4.7873.99 ms2; ln very low frequency (VLF), 4.9074.62 ms2 vs. 6.387 5.54 ms2; LF/HF ratio, 1.470.4 vs. 2.570.1]. The survival rate was similar between the groups. Conclusion: This study shows that OL-HDF improved autonomic nervous system dysfunction in chronic HD patients.