Abstract: BACKGROUND: There is little published data on how to prepare probiotic supplements for enteral delivery in the NICU. The objective of this study was to determine how a three-strain probiotic blend (Bb-02, TH-4® and BB-12®) would behave when mixed and held for 4 hours with saline water, sterile water, dextrose 5% in water (D5W), 24 kcal preterm formula, and human milk. METHODS: A packet of a three-strain probiotic supplement was mixed with 3 mL of saline water, sterile water, D5W, 24 kcal preterm formula, and human milk (tested at 3 mL and 2 mL). Samples were stored at room temperature for 60 minutes then refrigerated for 180 minutes. Probiotic survival, using quantitative enumeration, and pH were monitored over 4 hours. Samples were passed through a 5 French (Fr) feeding tube at the end of the study to evaluate viscosity. RESULTS: The largest variation in total cell count from 0-time was sterile water with a + 0.26 log(CFU)/mL change at 90 minutes and typical variation is considered±0.50 log units indicating no significant change between samples in 4 hours. Saline water had the lowest final pH at 4.88. All samples easily passed through a 5 Fr feeding tube. CONCLUSION: The study showed minimal change in cell counts across solutions for 4 hours of storage, indicating health care facilities may be able to prepare probiotic supplements with a variety of solutions in pharmacies or milk rooms. This allows greater flexibility for probiotic delivery to preterm infants.