Abstract: Frozen whole wheat bread can often cause the quality decline after reheating. This study aims to explore the effects of reheating modes on the gluten protein aggregation, eating quality, and flavor of the frozen whole wheat steamed bread. Bread samples with the different additions of the whole wheat flour were frozen at -18 ℃. A systematic comparison was also implemented to explore the effects of natural thawing, steam reheating, microwave reheating, and microwave steam reheating on the bread quality. The whole wheat bread was characterized by Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy, scanning electron microscopy (SEM), low field nuclear magnetic resonance (LF-NMR), and GC-IMS. The results showed that the steam recuperating energy effectively maintained the texture characteristics of frozen whole wheat bread, with the best hardness index (3 032.86 N). The structural characterization showed that the steam reheating treatment was significantly reduced the content of -SH in the gluten protein of whole wheat bread (P<0.05). The oxidation of free -SH in gluten protein also promoted to form the disulfide bonds. The elasticity and resilience of the product (P<0.05) were improved significantly after treatment. Natural thawing also promoted the protein aggregation behavior to maintain the electrostatic repulsion of gluten proteins. Hydrophobic analysis showed that the surface hydrophobicity of gluten protein was significantly improved (P<0.05), as the amount of whole wheat flour (WWF) added increased. The exposure of hydrophobic groups was exacerbated after reheating, due to the unstable gluten network structure caused by WWF during freezing; Among them, microwave reheating also exhibited low hydrophobicity, due to the short-term elevated temperature and starch gelatinization masking hydrophobic groups. While the steam reheating expanded the gluten network under hot steam, indicating the higher surface hydrophobicity. The scanning electron microscope showed that the exposure outside was represented for the starch particles in the whole wheat bread, naturally thawed after frozen storage. The gluten network and starch particles were wrapped more closely with each other after the steam reheating and microwave steam reheating, compared with microwave reheating. Grain size analysis showed that there was the a minimum grain size of gluten protein in the whole wheat bread of 0WWF, 50WWF, and 100WWF, which were 558.50, 592.86, and 615.30, respectively, under microwave reheating. The microwave steam composite treatment was reduced the conformational changes of the whole wheat proteins after the freezing. The fluorescence spectra of proteins showed the high fluorescence intensity in both microwave and steam reheating, indicating that microwave steam reheating was a relatively mild treatment. At the same time, the LF-NMR analysis showed that the high proportion of whole wheat was reduced the free water content. The mMore water existed in the bound state, indicating that microwave reheating and microwave steam reheating were conducive to the water quality of whole wheat bread. In addition, the flavor analysis confirmed that steam reheating shared the little effect on the whole wheat bread; Microwave reheating and microwave steam reheating were reduced the concentration of irritating flavor substances, such as the diethylene glycol dimethyl ether, benzaldehyde, ethylbenzene, butyraldehyde, and 2-hexanone, with the increasing concentration of fruity flavor substances, such as the propyl propionate and 3-methylethyl butyrate. The sensory evaluation showed that the microwave steam reheating effectively maintained the color, shape, and taste quality of whole wheat bread with the different amounts of whole wheat flour (0, 50, and 100WWF), especially when 100% whole wheat flour was added. There was the synergistic effect of microwave rapid internal heating and steam humid environment; Microwave reheating shared the higher scores in the appearance and odor dimensions, due to the heating speed. But the overall quality was still better in the microwave steam group. This finding can also provide an important theoretical basis to optimize the reheating process of frozen whole wheat flour products.