Aim: Repeated, ongoing exposure to pain influences the growth, cognitive and motor functions, behaviour, personality and neurodevelopment of preterm infants. We compared the analgesic effects of expressed breast milk (EBM) and 24% oral sucrose on preterm neonates during venipuncture.Methods: This multicentre randomised, noninferiority, crossover trial focused on five neonatal university units in Madrid, Spain, from October 2013 to October 2014. It comprised 66 preterm infants born at less than 37 weeks and randomly split into two groups. They received either EBM or sucrose two minutes before venepuncture, together with nonnutritive sucking and swaddling, then the opposite procedure at a later point. Pain was measured with the premature infant pain profile (PIPP) and crying was also measured.Results: There were no statistically significant differences between the groups. The PIPP scores were seven (4-9) with breast milk and six (4-8.25) with sucrose (p = 0.28). The 11 infants born at under 28 weeks of age showed higher median scores of nine (9-14) for breast milk and four (4-7) for sucrose (p = 0.009).Conclusion: EBM and 24% sucrose had the same analgesic effect during venipuncture in most of the preterm neonates, but sucrose worked better in extremely preterm infants.