PURPOSE: The aim of this study was to assess the various doses of oral oxybutynin on cardiac autonomic modulation by measuring short-term heart rate variability (HRV) indexes during supine rest position. METHODS: Eight male healthy subjects (20-23 years) participated in the double-blind crossover randomized study. The single dose of oxybutynin (2.5, 5 and 10 mg) or placebo was given to the volunteers in four sessions within 5-day intervals. Before and minutes of 30, 60, 90 and 120 after administration, lead II electrocardiogram (ECG) was recorded for 5 min. ECG extracted RR intervals data became the base of the calculation of time domain and frequency domain HRV parameters, which indicate cardiac autonomic activity. Statistical analysis was done by using the nonparametric Wilcoxon and Kruskal-Wallis tests. RESULTS: The data analysis has revealed that MNN (P < 0.001), SDNN (P < 0.05), PNN50% (P < 0.01), RMSSD (P < 0.001), HFnu (P < 0.05) and LF/HF ratio (P < 0.05) values were significantly increased relative to baseline at various time points in all the groups except in placebo group. LFnu (P < 0.05) values were significantly increased relative to baseline at various time points in all the groups except in placebo group. CONCLUSIONS: Our findings have revealed that acute consumption of 2.5, 5 and 10 mg oxybutynin (an anticholinergic compound) in the juvenile healthy male subjects produces a cholinergic effect according to time and frequency domain of HRV indexes.