AIMS: To determine the reproducibility and minimum detectable change (MDC) of heart rate variability (HRV) measures during two sequential 24-h periods, at week 32 of gestation, in preterm infants born between 28 and 32鈥痺eeks, hospitalized in the neonatal intensive care unit (NICU). The second aim is to assess postnatal changes in HRV measures between 32 and 35鈥痺eeks. STUDY DESIGN: 32 preterm infants born between 28 and 32鈥痺eeks of gestation were recruited. For each infant 48鈥痟 of recordings of RR interval were performed at week 32 and week 35. HRV parameters included time and frequency parameters. RESULTS: At week 32, the intra-class correlation coefficient (ICC) of all HRV values was statistically significant with high correlation coefficients (ICC鈥?鈥?.83-0.97). At week 35, a significant increase was noted in the HRV parameters, characterize mainly the sympathetic tone, with over half the infants showing an increase greater than the MDC for these parameters. CONCLUSIONS: Using 24-h recording at week 32 of gestation during NICU routine is reliable, feasible, not costly and may have important implications for an early identification of premature in a state of stress such as sepsis, or as a follow-up measure.