OBJECTIVE Systems controlling cardiovascular function are closely coupled with the perception of pain. Heart rate variability (HRV) is a well-established noninvasive measure of cardiac autonomic control. We hypothesized that pain may alter HRV in the newborn infant and that HRV analysis could be used as an indicator of prolonged pain in the newborn infant. METHODS To test the hypothesis, we measured the magnitude of the heart rate high-frequency variations using an innovative High Frequency Variability Index (HFVI) in newborn infants at risk of postoperative pain. We investigated newborn infants with a gestational age (GA) more than 34 weeks, and who were admitted after a major surgical procedure. Inclusions ranged from 2 to 72 hours after the surgery. The postoperative pain was scored using EDIN scale (neonatal pain and discomfort scale) at the end of the 2 hours recording period. The infants were separated in: (1) Group "Low EDIN," when EDIN<5; and (2) Group "High EDIN," when EDIN >=5. Predictive positive and negative values of a threshold value of HFVI in assessing pain have been studied. RESULTS Twenty-eight newborn infants were enrolled in the study (mean GA=37.8+/-1.5 wk) at a median delay between the surgery and the recording of 5 hours. Mean EDIN were 2+/-1 and 7+/-2 in respectively the groups "Low EDIN" and "High EDIN." The 2 groups were similar for GA, basal heart and respiratory rates, SpO2, mean arterial blood pressure, and morphine infusion rate. HFVI was significantly lower in the group "High EDIN" than in the group "Low EDIN" (0.7+/-0.2 vs. 1.2+/-0.3, respectively; P<0.01). An HFVI <0.9 was able to predict an EDIN score >=5, with a sensitivity of 90%, and a specificity of 75%. CONCLUSIONS The results of this study indicate that postoperative pain is associated with a decreased high-frequency HRV in full-term newborn infants. Our findings suggest that HRV could be used as an indicator to assess prolonged pain in the newborn infants.