BACKGROUND Despite the increasing interest in fetal and neonatal heart rate variability (HRV) analysis and its potential use as a tool for early disease stratification, no studies have previously described the normal trends of HRV in healthy babies during the first hours of postnatal life. METHODS We prospectively recruited 150 healthy babies from the postnatal ward and continuously recorded their electrocardiogram during the first 24 h after birth. Babies were included if born in good condition and stayed with their mother. Babies requiring any medication or treatment were excluded. Five-minute segments of the electrocardiogram (non-overlapping time-windows) with more than 90% consecutive good quality beats were included in the calculation of hourly medians and interquartile ranges to describe HRV trends over the first 24 h. We used multilevel mixed effects regression with auto-regressive covariance structure for all repeated measures analysis and t-tests to compare group differences. Non-normally distributed variables were log-transformed. RESULTS Nine out of 16 HRV metrics (including heart rate) changed significantly over the 24 h [Heart rate p < 0.01; Standard deviation of the NN intervals p = 0.01; Standard deviation of the Poincaré plot lengthwise p < 0.01; Cardiac sympathetic index (CSI) p < 0.01; Normalized high frequency power p = 0.03; Normalized low frequency power p < 0.01; Total power p < 0.01; HRV index p = 0.01; Parseval index p = 0.03], adjusted for relevant clinical variables. We observed an increase in several HRV metrics during the first 6 h followed by a gradual normalization by approximately 12 h of age. Between 6 and 12 h of age, only heart rate and the normalized low frequency power changed significantly, while between 12 and 18 h no metric, other than heart rate, changed significantly. Analysis with multilevel mixed effects regression analysis (multivariable) revealed that gestational age, reduced fetal movements, cardiotocography and maternal chronic or pregnancy induced illness were significant predictors of several HRV metrics. CONCLUSIONS Heart rate variability changes significantly during the first day of life, particularly during the first 6 h. The significant correlations between HRV and clinical risk variables support the hypothesis that HRV is a good indicator of overall wellbeing of a baby and is sensitive to detect birth-related stress and monitor its resolution over time.