Abstract The effect of frozen storage on the water sorption capability, water mobility, secondary structure, thermal and microscopic properties of gluten-, glutenin- and gliadin-rich fractions were investigated. Lower water sorption capability was observed for samples after frozen storage, suggesting that more hydrophobic moieties were exposed. Conversions of α-helix structure to β-turn structures and specific β-sheet structures were observed in the secondary structure analysis of gluten- and gliadin-rich fractions. Frozen storage induced higher water mobility in hydrated gluten proteins. Similar changes were observed in gluten-water and gliadin-water systems, implying that the changes were primarily attributed to subdued gliadin-water interactions and gliadin can stabilize glutenin network to confine the water mobility. Meanwhile, thermo gravimetric analysis (TGA) and differential scanning calorimetry (DSC) showed that thermal degradation temperature decreased while thermal denaturation stability increased in gluten- and glutenin-rich fractions with the increasing time of frozen storage. However, the enthalpies of all the gluten proteins decreased, indicating more disordered structures in the aged gluten proteins. The micrographs of scanning electron microscopy (SEM) also confirmed more disordered and weak structures in gluten- and glutenin-rich fractions induced by frozen storage. Furthermore, consistent changes in gluten-, glutenin- and gliadin-rich fractions indicated that the variations in conformational, thermal and microscopic properties of gluten might originate from glutenin and gliadin upon frozen storage.