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The effects of water and glycerol on the molecular mobility of a soy-protein isolate (SPI)-based film were investigated using 1H nuclear magnetic resonance (NMR). SPI-films were prepared with different glycerol contents and equilibrated to various levels of water activity (aw). The distributed transverse relaxation of film samples showed 2 distinct components: a solid-like fraction (T2s*) and a liquid-like fraction (T2m*). The peak T2s* values of films with lower glycerol concentration were more sensitive to variation in aw. As aw increased, the change of solid-like to liquid-like fraction was observed in higher glycerol films. As glycerol content increased, a minimum longitudinal relaxation time (T1) occurred at lower aw. Our results suggest that NMR techniques can be used to quantify the plasticizing effects on biopolymer films.