Sinus node dysfunction (SND) causes significant morbidity in patients after Fontan surgery. Heart rate variability (HRV) reflects the autonomic regulation of the heart, and changes in HRV have been associated with SND in adults. We aimed to study whether changes in HRV could be detected in 24-h electrocardiographic (ECG) recordings in Fontan patients with SND. We compared HRV results from two patient groups; patients with Fontan circulation who later required a pacemaker due to severe SND ( n = 12) and patients with Fontan circulation and SND, without indication for pacemaker treatment ( n = 11), with two control groups; patients with Fontan circulation without SND ( n = 90) and healthy controls ( n = 66). The Poincaré plot index SD2 (representing changes in heart rate over 24-h) and the very low-frequency (VLF) HRV component were significantly higher in both SND groups, both compared with healthy controls and patients with Fontan circulation without SND. In SND patients with pacemakers, SD2 and VLF were slightly reduced compared to SND patients without pacemaker ( p = 0.06). In conclusion, in Fontan patients with SND the HRV is significantly higher compared to healthy controls and Fontan patients without SND. However, in patients with severe SND requiring pacemaker, SD2 and VLF tended to be lower than in patients with SND without pacemaker, which could indicate a reduced diurnal HRV in addition to the severe bradycardia. This is a small study, but our results indicate that HRV analysis might be a useful method in the follow-up of Fontan patients regarding development of SND.