To determine the effect of prone sleeping on cerebral oxygenation in preterm infants in the neonatal intensive care unit.Preterm infants, divided into extremely preterm (gestational age 24-28 weeks; n = 23) and very preterm (gestational age 29-34 weeks; n = 33) groups, were studied weekly until discharge in prone and supine positions during active and quiet sleep. Cerebral tissue oxygenation index (TOI) and arterial oxygen saturation (SaO) were recorded. Cerebral fractional tissue extraction (CFOE) was calculated as CFOE = (SaO - TOI)/SaO.2In extremely preterm infants, CFOE increased modestly in the prone position in both sleep states at age 1 week, in no change in TOI despite higher SaO. In contrast, the very preterm infants did not have position-related differences in CFOE until the fifth week of life. In the very preterm infants, TOI decreased and CFOE increased with active sleep compared with quiet sleep and with increasing postnatal age.2At 1 week of age, prone sleeping increased CFOE in extremely preterm infants, suggesting reduced cerebral blood flow. Our findings reveal important physiological insights in clinically stable preterm infants. Further studies are needed to verify our findings in unstable preterm infants regarding the potential risk of cerebral injury in the prone sleeping position in early postnatal life.