Extremely premature infants receive nutrition and medication through nasogastric tubes. Breastmilk given accordingly is subject to fat loss. This study aimed to investigate whether this could also apply to vitamin D. A questionnaire investigated vitamin D administration at a level III neonatal intensive care unit in Sweden in 2021. Feeding simulations with breastmilk and various vitamin D mixtures were done accordingly. After administration, vitamin D3 concentration was analysed using chromatography with mass spectrometry, followed by repeated simulations with vitamin D mixtures without breastmilk in 2023. The questionnaire was completed by 10 persons. Vitamin D was administered as drops using an enteral syringe and a nasogastric tube in conjunction with a breastmilk meal. In the feeding simulations, vitamin D3 concentration after administration was significantly higher using a syringe alone compared to standard administration. When vitamins were administered according to standard but without breastmilk, 100% of the vitamin D and 40% of the multivitamins were lost. The vitamins adhered to the material, mainly in the nasogastric tube. Our findings indicate that standard vitamin D supplementation in the neonatal intensive care unit may be unpredictable when administered by enteral syringe and nasogastric tube. We suggest using direct oral administration whenever possible.