PURPOSE: Modulation of the intrinsic cardiac autonomic nervous system (ICANS) has been described to occur during radiofrequency pulmonary vein (PV) isolation for paroxysmal atrial fibrillation (AF) and has been controversially proposed to increase clinical success. Energy source used for PV isolation might influence ICANS modulation. The effect of balloon-delivered cryoenergy on the ICANS is unknown. We conducted a study investigating acute periprocedural effect on ICAN as well as changes in heart rate variability (HRV) for standard deviation of normal-to-normal intervals (SDNN) and triangular index (TI) as surrogates for ICANS modulation after cryoballoon PV isolation. METHODS: Fourteen consecutive patients without structural heart disease underwent cryoballoon PV isolation for paroxysmal atrial fibrillation. Acute changes in heart rate requiring pacing during the procedure were recorded. HRV was tested by Holter ECG for SDNN and TI before ablation and after 1 week, 1 month, and 3 months following ablation. RESULTS: Fifty-five out of 56 PV were isolated (98%) with short-term 6-month freedom from paroxysmal AF of 64% by one single procedure. Five patients (36%) showed significant bradycardia during balloon thawing requiring temporary pacing. HRV decreased significantly immediately after PV isolation for both SDNN and TI until 1 month, gradually normalizing toward 3 months follow-up. HRV modulation was not different between patients with or without AF recurrences. CONCLUSIONS: Cryoballoon PV isolation significantly modulates the ICANS, but only temporarily for up to 3 months, measured by HRV changes after ablation.