The objective of this study was to investigate the effect of EMLA on the pain response when venipuncture was performed in 60 3-day-old healthy newborns. EMLA/placebo was applied to the back of the baby's hand, following a randomized, double-blind procedure. ECG and crying were recorded during the test. The incidence of crying, heart rate (HR) and spectral analysis of heart rate variability were used to characterize the reaction of the baby to the venipuncture procedure. The occurrence of crying during venipuncture did not differ significantly between the EMLA and the placebo groups. The placebo-treated group showed a statistically significant higher HR, and a decrease in variance (total power) and power in the low-frequency band (0.02-0.15 Hz) when compared with the EMLA group. We conclude that EMLA decreases the stress response during venipuncture in newborn infants.