The aim of this randomized crossover study was to evaluate the impact of environmental and behavioral interventions (EBI) on behavioral, physiologic, and biologic stress response during a weighing procedure in neonates. Three groups of 15 neonates included (A) gestational age (GA), < or =32 weeks; (B) GA, 32 weeks, 1 day to 36 weeks, 6 days; and (C) GA, > or =37 weeks. Each neonate experienced 2 weighing procedures with and without EBI. Pain was evaluated by using the Neonatal Infant Pain Scale (NIPS) and the Neonatal Pain and Discomfort Scale (EDIN). Heart rate and oxygen saturation were recorded. Salivary samples were obtained for cortisol assay. Cerebral tissue oxygenation index (TOI) was recorded with near-infrared spectroscopy. A significant decrease of NIPS and EDIN was observed with EBI versus control. Mean heart rate was lower with EBI. No difference in cortisol level changes was observed. For groups A and B, a trend of increased TOI was observed with EBI. We concluded that EBI during a nursing procedure provides a decrease in pain scores in preterm and term neonates with changes in heart rate. PERSPECTIVE: This study evaluates the impact of combined environmental and behavioral interventions on pain responses in neonates during a weighing procedure. The results indicate a decrease in behavioral pain scores and in heart rate for preterm and term neonates and a trend in increased brain oxygenation depending on gestational age.