Aim: Anticholinergic drugs may alter cardiac autonomic modulation. The aim of this study was to investigate the effects of inhaled tiotropium on heart rate variability (HRV). Methods: A randomized, double-blind, crossover design study was conducted on 11 healthy volunteers. Tiotropium or placebo was administered in two different testing sessions. Time domain parameters; mean R-R interval (mean-RR), the standard deviation of R-R interval (SDNN) and the root mean square of successive R-R interval differences (RMSSD) and power spectral analysis of HRV were assessed in the supine position, during handgrip exercise and controlled breathing before and after drug administration. Results: There were no statistically significant difference in baseline HRV parameters obtained before each drug administration (p >0.05). HRV parameters obtained after each drug administration were found not to be different during supine position, controlled breathing and handgrip exercise. Conclusion: Administration of single dose tiotropium did not effect cardiac autonomic modulations in healthy volunteers.