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Objective To examine the effects of 30% oral dextrose on biochemical markers of pain, adenosine triphosphate (ATP) degradation, and oxidative stress in preterm neonates experiencing a clinically required heel lance. Study design Utilizing a prospective study design, preterm neonates that met study criteria (n = 169) were randomized to receive either (1) 30% oral dextrose, (2) facilitated tucking, or (3) 30% oral dextrose and facilitated tucking 2 min before heel lance. Plasma markers of ATP degradation (hypoxanthine, uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the premature infant pain profile-revised (PIPP-R). Results Oral dextrose, administered alone or with facilitated tucking, did not alter plasma markers of ATP utilization and oxidative stress. Conclusion A single dose of 30% oral dextrose, given before a clinically required heel lance, decreased signs of pain without increasing ATP utilization and oxidative stress in premature neonates.