Objective Although many perinatal factors have been linked to adverse neurodevelopmental outcomes in very premature infants, much of the variation in outcome remains unexplained. The impact on brain development of one potential factor, exposure to stressors in the Neonatal Intensive Care Unit, has not yet been studied in a systematic, prospective manner. Methods In this prospective cohort study of infants born at <30 weeks gestation, nurses were trained in recording procedures and cares. These recordings were used to derive Neonatal Infant Stressor Scale scores, which were employed to measure exposure to stressors. Magnetic resonance imaging (brain metrics, diffusion, and functional magnetic resonance imaging) and neurobehavioral examinations at term equivalent postmenstrual age were used to assess cerebral structure and function. Simple and partial correlations corrected for confounders including immaturity and severity of illness were used to explore these relationships. Results Exposure to stressors was highly variable, both between infants and throughout a single infant’s hospital course. Exposure to a greater number of stressors was associated with decreased frontal and parietal brain width, altered diffusion measures and functional connectivity in the temporal lobes, and abnormalities in motor behavior on neurobehavioral examination. Interpretation Exposure to stressors in the Neonatal Intensive Care Unit is associated with regional alterations in brain structure and function. Further research into interventions that may decrease or mitigate exposure to stressors in the Neonatal Intensive Care Unit is warranted.