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The effects of starch and gluten on the physicochemical properties of frozen dough were studied using reconstituted flour. The profiles of frozen dough were studied by Mixolab, rheometer, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR). Results revealed that starch, rather than gluten, played a decisive role in mixing properties. The breakdown and aggregation of the gluten network structure as well as the formation of β-turns and β-sheets in the frozen dough would be aggravated by the freezing of wheat starch. Smaller wheat starch granules (B-Type granules) affected the secondary structure of gluten network more than larger granules (A-Type granules), resulting in greater rheological property changes. The viscoelastic properties and freezable water content of frozen dough were more influenced by the freezing of gluten. [Display omitted] • Starch had more impact on mixing characteristics than gluten in frozen dough. • Frozen wheat starch would aggravate the disintegration and aggregation of gluten. • B-Type granules affected the secondary structure of gluten more than A-Type granules. • Gluten played a more important role in the viscoelastic properties of frozen dough. [ABSTRACT FROM AUTHOR]