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Oral sucrose is administered routinely to reduce pain of minor procedures in premature infants and is recommended as standard care in international guidelines. No human or animal studies on effects of early repeated sucrose exposure on long-term brain development have been done in the context of pain. We evaluated the effects of repeated neonatal sucrose treatment before an intervention on long-term brain structure in mouse pups. Neonatal C57Bl/6J mice (n = 109) were randomly assigned to one of 2 treatments (vehicle vs sucrose) and one of 3 interventions (handling, touch, or needle-prick). Mice received 10 interventions daily from postnatal day 1 to 6 (P1-6). A dose of vehicle or 24% sucrose was given orally 2 minutes before each intervention. At P85-95, brains were scanned using a multichannel 7.0 T MRI. Volumes of 159 independent brain regions were obtained. Early repetitive sucrose exposure in mice (after correcting for whole brain volume and multiple comparisons) lead to smaller white matter volumes in the corpus callosum, stria terminalis, and fimbria (P < 0.0001). Cortical and subcortical gray matter was also affected by sucrose with smaller volumes of hippocampus and cerebellum (P < 0.0001). These significant changes in adult brain were found irrespective of the type of intervention in the neonatal period. This study provides the first evidence of long-term adverse effects of repetitive sucrose exposure and raises concerns for the use of this standard pain management practice during a period of rapid brain development in very preterm infants.