Pyridostigmine bromide (PB) is a reversible cholinesterase inhibitor. The aim of this study was to determine the effect of orally administration of single dose sustained-released tablet of pyridostigmine bromide (PBSR) on the frequency domain indices of heart rate variability (HRV). Thirty-two healthy young men were participated in this study. They were divided into 2 groups; the pyridostigmine group (n = 22) and the placebo group (n = 10). Electrocardiogram (ECG) was recorded at 10, 30, 60, 90, 120, 150, 180, 210, 240, 300 and 420 min after PBSR administration. At each time, simultaneously, a blood sample was prepared and PB plasma concentration was measured by high-performance liquid chromatography (HPLC) method. Statistical analysis showed that in different indices of HRV, there is a significant increase in low frequency (LF) band at 300 min, but no difference in high frequency band (HF). It also showed significant decreases in normalized high frequency band (Hfnu), normalized low frequency band (Lfnu) and LF/HF ratio at 120, 240 and 300 min after PBSR administration. Maximum plasma concentration of PB was 150 min after the administration. In conclusion, administration of a single dose PBSR can enhance the frequency domains indices of HRV and improvesympathovagal balance.