Objective: To determine whether human milk fortification from the time of the first feeding significantly improves weight gain and bone mineral status in infants of <31 weeks estimated gestational age as compared with delayed or standard human milk fortification.Study Design: This was a retrospective pre-post design. In all, 95 infants born at <31 weeks estimated gestational age were compared. There were 53 infants in the early fortification group (EFG) and 42 infants in the delayed fortification group (DFG). They were compared with regard to weight gain at 34 weeks postmenstrual age (PMA), and their serum levels of calcium, phosphorus and alkaline phosphatase levels were compared as an indicator of bone mineral status. The practice change of fortifying all human milk given to preterm infants at <34 weeks PMA commenced in June 2009. The usual practice of fortification took place once an infant had reached a feeding volume of 50 to 100 ml kg À1 per day. The new practice fortified all human milk with a powdered human milk fortifier to 24 calories per ounce, starting with the first feeding, no matter how small the volume.Result: There were no differences in weight gain between the EFG and the DFG. The group that received fortification from the time of the first feeding were significantly less likely to have alkaline phosphatase levels >500 U l À1 from 33 weeks PMA onward. There was no incidence of feeding intolerance with early fortification.Conclusion: Fortification of human milk from the time of the first feeding does not affect weight gain at 34 weeks PMA, but is related to a lower incidence of elevated alkaline phosphate levels and does not cause feeding intolerance.