The time course and concentration-effect relationship of parasympatholytic effects of three anticholinergic drugs were investigated using spectral analysis of heart rate (HR) variability. Single intravenous (IV) doses of atropine (10 µg/kg), glycopyrrolate (5 µg/kg), scopolamine (5 µg/kg), and placebo were given to eight healthy volunteers in a double-blind, randomized cross-over study. Electrocardiogram (ECG) was recorded at baseline and 2.5, 5, 10, 20, and 30 minutes, and 1, 1.5, 2, 3, 4, 5, and 6 hours after drug administration, while the subjects breathed at a fixed 0.25 Hz frequency. The powers of two frequency bands (low frequency [LF] = 0.07-0.15 Hz and high frequency [HF] = 0.15-0.40 Hz) were calculated using stationary time series of R-R intervals (RRI) free from ectopic beats. To perform pharmacokinetic-pharmacodynamic (PK-PD) modeling, venous plasma drug concentrations were measured. Atropine and glycopyrrolate, and, to a lesser extent, scopolamine induced decreases in HF power and increases in LF/HF ratio of HR variability, indicating parasympatholytic activity and corresponding changes in sympathovagal balance. Maximal average decreases in HF power were 99%, 94%, and 82%, respectively, but in two scopolamine subjects, a parasympathomimetic effect was dominant. Interindividual variability was least for the Hayano index of HF power (√ (RRI HF-power)/RRI*100), and profound and consistent decreases were seen after atropine and glycopyrrolate. Pharmacokinetics were best fitted to a two-compartment open model, and effect compartment link modeling using the Hayano index was performed with the atropine and glycopyrrolate data. The best description of the PK-PD relationship for both drugs was achieved using the sigmoidal Emaxmodel. Mean (±SD) EC50, sigmoidicity factor (γ), and equilibration rate constant (keO) estimates were 1.35 (±0.27) ng/mL, 6.07 (±1.98) and 11.0 (±5.28) 1/h for atropine and 1.35 (±0.49) ng/mL, 4.34 (±1.55) and 2.26 (±0.81) 1/h for glycopyrrolate. Spectral analysis of HR variability appears to be a powerful tool in monitoring parasympatholytic drug activity. A sigmoidal Emaxmodel with an extremely steep concentration-response relationship was revealed for atropine and glycopyrrolate. The effects of scopolamine were more incongruous.