ObjectiveTo investigate the effects of breast milk on cortical pain response and behavioral response in newborns during heel-prick procedure.Study designA prospective, randomized controlled trial was conducted on healty-term newborns, undergoing heel blood sampling. Infants were randomly assigned to study group with receive orally 2 ml breast milk (n = 45) or a control group with no intervention (n = 45). A near-infrared spectroscopy device was used to monitor regional cerebral oxygen saturation (rScO2), while neonatal pain expression was assessed by Neonatal Pain, Agitation, and Sedation Scale (N-PASS).ResultsThe N-PASS score (p = 0.001) and the crying time (p = 0.017) were significantly lower in the study group compared to the control group. Although the mean rScO2 values decreased in both groups during the procedure, the percent change in rScO2 was not significant difference between two groups.ConclusionBreast milk administration decreases behavioral responses to a noxious stimulus without reducing the cortical response to pain.Clinical trial registrationThis trial was registered under ClinicalTrials.gov identifier no. NCT05961904.