The biochemical conversion of lignocellulosic biomass into renewable fuels and chemicals provides new challenges for industrial scale processes. One such process, which has received little attention, but is of great importance for efficient product recovery, is solid-liquid separations, which may occur both after pretreatment and after the enzymatic hydrolysis steps. Due to the changing nature of the solid biomass during processing, the solid-liquid separation properties of the biomass can also change. The objective of this study was to show the effect of enzymatic hydrolysis of cellulose upon the water retention properties of pretreated biomass over the course of the hydrolysis reaction. Water retention value measurements, coupled with H-1 NMR T-2 relaxometry data, showed an increase in water retention and constraint of water by the biomass with increasing levels of cellulose hydrolysis. This correlated with an increase in the fines fraction and a decrease in particle size, suggesting that structural decomposition rather than changes in chemical composition was the most dominant characteristic. With increased water retained by the insoluble fraction as cellulose hydrolysis proceeds, it may prove more difficult to efficiently separate hydrolysis residues from the liquid fraction with improved hydrolysis.