Background: Serum bilirubin levels beyond the physiological limits, may lead to alterations in autonomic regulation in a newborn infant. Heart rate variability (HRV), is a noninvasive and quantitative marker of the activity of the autonomic nervous system (ANS). To date, few studies have demonstrated the undesirable effects of severe unconjugated hyperbilirubinemia (UHB) on autonomic functions, and only one study has used HRV as a marker of the autonomic activity. However, the relationship between altered cardiac autonomic functions and UHB by using the HRV derived from 24-hour Holter electrocardiography (ECG) recording has not been investigated previously.Objective: We aimed to assess whether a relationship exists between severe UHB and cardiac autonomic dysfunction by evaluating HRV via 24-hour Holter ECG recording.Methods: This single-center, prospective, case-control study was conducted on 50 full-term newborn infants with severe UHB requiring phototherapy and 50 healthy infants as controls. HRV assessment was performed by using 24-hour Holter ECG recording.Results: There was no significant difference in terms of mean average heart rate, mean maximum heart rate and mean RR duration between the groups. However, mean minimum heart rate was significantly lower in the study group. When 24-hour time and frequency domain parameters were compared, time and frequency domain parameters rMSDD as well as high frequency (HF), which represent parasymphathetic activity, were significantly higher in the study group. Furthermore, low frequency to high frequency (LF/HF) ratio, that serves as an indicator of sympathovagal balance, was significantly lower in the study group.Conclusion: Severe UHB may cause cardiac autonomic dysfunction in favor of parasympathetic predominance in jaundiced neonates.