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The efficacy of oral sucrose in the reduction of single episodes of acute procedural pain in newborn infants has been demonstrated in a large number of well-conducted randomized controlled trials. However, there are few studies that have examined the effectiveness of repeated doses of sucrose and there are no studies of prolonged sucrose use in sick infants over an entire period of hospitalization.The aim of this study was to evaluate the analgesic effectiveness of repeated doses of oral sucrose during heel lancing in sick infants over the course of a prolonged hospitalization.A prospective longitudinal cohort study was conducted in a tertiary referral neonatal intensive care unit. Infants with a predicted length of stay of ≥28 days were eligible for inclusion. Oral sucrose was administered prior to and during all heel lance procedures observed. Pain outcomes consisted of facial expression scores, crying duration, heart rate, and oxygen saturation. Changes in sequential pain responses were analyzed, and weighted linear regression slopes for all assessed parameters were calculated to estimate the average regression slope.There were 55 infants enrolled, and 443 pain assessments during heel lancing were conducted. Behavioral responses to heel lancing were predominantly low, and there were minimal changes in behavioral and physiological parameters to successive heel lance procedures. The mean weighted regression slope over the period of hospitalization for the facial expression scores upon heel lance, on the 0-4 scale, was −0.0084 (SD = 0.298; 95% confidence interval [CI] = −0.076 to 0.093; p = .84). The mean weighted regression slope for crying proportion during the procedure was −0.046% (SD = 4.247%; 95% CI = −1.322% to 1.230%; p = .943) and for heart rate change from baseline during the heel lance procedures was 0.541% (SD = 2.907%; 95% CI = −0.299% to 1.353%; p = .02).The predominantly low behavioral responses to heel lancing and the lack of increase in behavioral pain outcomes suggest the ongoing effectiveness of oral sucrose during painful procedures throughout the infants' hospitalization. Further studies are recommended to ascertain the influence of factors such as adjunct analgesics, sedatives, and severity of illness.