To investigate the effects of frozen storage on the rheological and microstructural properties of gluten, two model systems were investigated: System A, gluten and water; System B, gluten, water, and NaCl. The storage time was varied from 1 to 16 weeks and the storage temperature was varied from -5 to -30°C. After thawing, uniaxial and biaxial deformations, and stress relaxation measurements were performed on gluten. In System A, the major effects were noticed when the gluten was stored at -5°C. Frozen storage induced a decrease in stress and in strain at breaking, but an increase in modulus (stress/strain) under uniaxial deformation. In System B, only stress relaxation measurements showed differences between the fresh gluten and the gluten stored at -18°C. These results suggest that at -5°C, gluten strands form more entanglements. Microscopic analyses of frozen gluten showed that, during the freezing step, ice crystals compressed the gluten. A significant phase separation was observed between gluten and ice but no difference was observed between the storage time and storage temperature. However, after thawing, gluten microstructure exhibited a structure similar to the fresh gluten, and the structure looks like a sponge (a fine gluten structure with tiny water pockets).