The DiO probe was included in all lipid containing GUV compositions (0-75 mol% polymer) and is assumed to be representative of lipid diffusion in a hybrid membrane. The DiO probe diffusion coefficient decreased with increasing polymer fraction from 3.31 ± 0.56 µm s -1 for single component POPC GUVs (0 mol% polymer) to 0.46 ± 0.15 µm s -1 in 75 mol% PBd22-PEO14 hybrid vesicles and 0.68 ± 0.23 µm s -1 in 75 mol% PBd12-PEO11 hybrid vesicles. In PBd22-PEO14 vesicle compositions, only the reduction in DiO diffusion between 0 mol% and 25 mol% PBd22-PEO14 is statistically significant in nearest neighbour compositions. However, next-nearest neighbour and wider spread compositions did show statistically significant differences (p < 0.05, Tukey and Bonferroni ANOVA) in diffusion coefficient (comparing 0 mol% polymer with 50 and 75 mol% vesicles and 25 mol% with 75 mol% vesicles). For all PBd12-PEO11 hybrid vesicle compositions, the differences in DiO diffusion is statistically significant (p < 0.05). The large, significant drop in DiO diffusion coefficient between 0 mol% and 25 mol% PBd22-PEO14 GUVs suggests that a minority component of these larger polymers in hybrid membranes can have a dominant influence on the fluidity of the composite membrane.