Vulnerable preterm infants experience repeated and prolonged pain/stress stimulation during a critical period in their development while in the neonatal intensive care unit (NICU). The contribution of cumulative pain/stressors to altered neurodevelopment remains unclear. The study purpose was to investigate the impact of early life painful/stressful experiences on neurobehavioral outcomes of preterm infants in the NICU.A prospective exploratory study was conducted with fifty preterm infants (28 0/7-32 6/7weeks gestational age) recruited at birth and followed for four weeks. Cumulative pain/stressors (NICU Infant Stressor Scale) were measured daily and neurodevelopmental outcomes (NICU Network Neurobehavioral Scale) were examined at 36-37weeks post-menstrual age. Data analyses were conducted on the distribution of pain/stressors experienced over time and the linkages among pain/stressors and neurobehavioral outcomes.Preterm infants experienced a high degree of pain/stressors in the NICU, both in numbers of daily acute events (22.97±2.30 procedures) and cumulative times of chronic/stressful exposure (42.59±15.02h). Both acute and chronic pain/stress experienced during early life significantly contributed to the neurobehavioral outcomes, particularly in stress/abstinence (p<0.05) and habituation responses (p<0.01), meanwhile, direct breastfeeding and skin-to-skin holding were also significantly associated with habituation (p<0.01-0.05).Understanding mechanisms by which early life experience alters neurodevelopment will assist clinicians in developing targeted neuroprotective strategies and individualized interventions to improve infant developmental outcomes.