In this prospective study, the effects of mediastinal radiation therapy (RT) on autonomic nervous system (ANS) were investigated by heart rate variability (HRV) analysis that is accepted as a non-invasive indicator of ANS. Study was performed with the eligible patients had a histopathologically confirmed diagnosis of malignant disease with no known congestive heart failure, coronary heart disease, hypertension, valvular cardiac disease or arrhythmia history. Electrocardiograms of 14 voluntary patients were recorded for duration of 5 min just before and after irradiation for the first and the 15th fractions. ANS-related HRV analysis parameters were calculated as which were recommended by Task Force of ESC/NASPE (Circulation 93:1043-1065, 1996). HRV parameters that belong to pre- and post-RT treatment of patients were compared statistically. We found that there is not effect of single-dose radiation on HRV parameter. The mean RRI (782.29 ± 115.65-738.93 ± 111.01, P < 0.014) and HF power of HRVs PSD (156.94 ± 229.37-60.71 ± 77.99, P < 0.045) decreased, and LF/HF ratio (1.38 ± 0.79-2.03 ± 1.25, P < 0.039) increased significantly with 28-Gy external radiation dose. As the effect of cumulative dose was investigated on HRV parameters, the above changes were continued to increase with 30-Gy external radiation dose. We found that mediastinal RT involving heart directly in the radiation field decreased vagal and sympathetic ANS activities, and autonomic balance shifted toward sympathetic dominance.