Varenicline is an alpha4beta2 nicotinic acetylcholine receptor partial agonist. In this study, we assessed the effects of varenicline on heart rate variability (HRV). Thirty subjects were included in the randomized, double-blind, placebo-controlled, crossover study. Varenicline or placebo was administered in two different testing sessions. Time domain parameters and power spectral analysis of HRV were assessed in the supine position and during handgrip exercise before and after the participants were given placebo or varenicline. Fifteen healthy non-smokers (NS) and fifteen healthy smokers (S) were included in the study. There were no statistically significant differences among any of the time domain parameters obtained before and after placebo administration or between the S and NS groups with respect to varenicline administration. In frequency domain analyses, normalized HF (high-frequency) powers were significantly higher in the S group than in the NS group (before placebo, NS:6.57+/-3.58 vs. S:13.85+/-7.50, p=0.002, after placebo, NS:6.33+/-3.89 vs. S:10.82+/-4.88, p=0.007). After varenicline administration, the normalized HF power was significantly higher (NS:6.65+/-4.34 vs. S:11.06+/-4.52, p=0.01), and the ratio of LF (low-frequency) to HF was significantly lower (NS:8.44+/-5.89 vs. S:4.97+/-4.60, p=0.02) in the S group than in the NS group. Administration of a single dose of varenicline significantly increased the LF/HF ratio (5.83+/-2.69 vs. 8.44+/-5.89) in the NS group, but in the S group, there were no significant differences related to any of the time or frequency domain parameters. We concluded that a single dose of varenicline does not affect HRV in healthy smokers but that it may alter HRV when administered at a therapeutic dose to healthy non-smokers during mild sympathetic stimulation.