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Through Thick and Thin: The In Vitro Effects of Thickeners on Infant Feed Viscosity. BACKGROUND: Gastroesophageal reflux disease (GERD) is a common problem in neonates, and current modalities for thickening human milk produce inconsistent outcomes. The objective of this in vitro study is to measure the viscosity effect of different thickening strategies. METHODS: We thickened donor human milk (DHM) and formula using various thickeners: starch-based thickeners (SBT; Thick It, rice cereal), and gum-based thickeners (GBT; xanthan gum: Simply Thick, Thicken Up Clear; carob gum: GelMix). We also assessed formula with added starches marketed for reflux, including Similac Spit Up (SSU) and Enfamil AR (EAR). The viscosity of each sample was measured over time using a rotary viscometer. Additional variables, including acidity, temperature, and the addition of human milk fortifier, were tested. RESULTS: Formula can be effectively thickened with all tested thickeners, but the viscosities of thickened formula increase over time. On the other hand, DHM does not effectively thicken with SBT. Autoclaving DHM inactivates digestive enzymes, thus allowing SBT to successfully thicken autoclaved DHM. GBT effectively thickened both DHM and formula but reached higher viscosities than intended based on manufacturer recommendations. Adding acid to xanthan-gum thickened DHM resulted in phase separation and formation of solid precipitant. CONCLUSIONS: Current thickening strategies of preterm infant feeding produces highly variable results in final feed viscosity. The unpredictable properties of gum-based thickeners raise questions about their safety profile. Objective measures of liquid viscosity and careful consideration of acidity and time are recommended for adequate comparisons of thickening regimens. Human milk continues to be the most challenging feed type to thicken.