BACKGROUND: Complete isolation of the posterior left atrium, including all pulmonary veins (box isolation), is a feasible and safe atrial fibrillation (AF) treatment method. Data on long-term effects of box isolation on autonomic function, however, remain limited. METHODS AND RESULTS: A total of 92 patients (paroxysmal AF, n=76; persistent AF, n=16) undergoing box isolation with non-contact mapping were studied. Twenty-four-hours ambulatory electrocardiograms and transthoracic echocardiography were done at baseline and after 2 days, and at 3, 6 and 12 months. Autonomic functions were evaluated by means of heart rate variability (HRV). During a mean follow up of 16+/-5 months, no AF episodes were detected in 76 patients while AF recurred in 16. Significant long-term HRV attenuations were observed in all patients without AF recurrence, but not in those with AF recurrence. In patients without AF recurrence, the natural logarithm (Ln) high-frequency (HF) decreased significantly from 5.5+/-1.3 ms(2) (before) to 4.2+/-0.9 ms(2) (after 12 months, P<0.001) and the ratio of the low-frequency to HF power increased significantly from 2.4+/-2.0 (before) to 3.4+/-2.3 (after 12 months, P=0.05). LnHF was significantly lower in patients without than in those with AF recurrence during the 12-month period after ablation. Cardiac function improved significantly in patients without AF recurrence after box isolation. CONCLUSIONS: Changes in the sympathovagal balance with box isolation may contribute to AF prevention.