Objective: Preterm infants usually have low retinol status. We have therefore evaluated a new protocol designed to improve vitamin A status in very low birth weight infants (VLBW, birth weight < 1,500 g). Since the new protocol also changed the supplementation of vitamins D, E and K, their plasma concentrations were also analyzed to characterize the infants’ status of all fat-soluble vitamins.Design: An open intervention trial was conducted where vitamin A was given in a human milk fortifier. The daily intake of vitamin A was increased by 8% compared to the previous regimen (reference), and given mixed with human milk instead of as a bolus. In this modified regimen, the intake of the other fat-soluble vitamins also changed due to different vitamin content in the supplementation used. Vitamin D intake was reduced by 10%; vitamin E intake was reduced by 6% in the modified protocol and vitamin K intake was increased by 132% compared to the reference regimen. Blood samples were collected at inclusion and at discharge from hospital. The plasma concentrations of the different vitamins were analyzed with high performance liquid chromatography. The daily intake of all the fat-soluble vitamins and their plasma concentrations were compared to the vitamin protocol normally used in Norwegian hospitals.Results: Sixty VLBW infants were included and 53 completed the study. At discharge from hospital, the reference group had lower plasma retinol concentrations compared to the modified group (0.30 vs. 0.45 µM, P=0.005). Fewer infants in the modified group had plasma retinol levels below 0.35 µM (indicating reduced hepatic stores) compared to the infants in the reference group (44% vs. 69%, P = 0.04). At discharge the reference group had higher plasma 25(OH)vitamin D than the modified group (171 vs. 110 μM, P=0.003). The plasma concentrations of -tocopherol were not significantly different between the two groups (31 vs. 39 μM) at discharge from hospital. The plasma phylloquinone concentration was non-significantly lower in the reference group compared to the modified group (1.0 vs. 2.1 ng/ml).Conclusions: The modified regimen improved vitamin A status among VLBW infants at discharge compared to the reference protocol. More studies are needed to establish the optimal plasma retinal concentration for preterm infants and how this can be achieved. Whether this will translate into lower vitamin A-related morbidity and/or mortality in these children warrants further investigation. Vitamin D status was also improved in the modified group by giving a plasma concentration in the normal range compared to the high mean level in the reference group. Vitamin E status was similar in the two groups and vitamin K status was not significantly improved.