Abstract: Vacuum mixing is a new method of dough mixing in noodle industrial production which allows more water in the noodle formula and facilitates gluten development. The quantity and molecular structure of glutenin macropolymer (GMP) governs the gluten strength and dough properties, especially in flour products' processing. In order to learn about the changes of GMP characteristics under vacuum mixing, 3 wheat cultivars with different quality were selected as the material, and mixing experiments were conducted under different vacuum levels (0, 0.06 and 0.08 MPa). GMP content and particle size distributions of freeze-dried noodle doughs were determined, and the free sulfhydryl group (-SH) contents in total protein and GMP were measured respectively using Ellman reagents. The results showed that the vacuum mixed noodle dough at 0.06 MPa had a higher GMP content compared with the non-vacuum mixed dough. However, extremely high vacuum degree (0.08 MPa) could decrease the GMP content. Vacuum mixing at an appropriate vacuum level induced a decrease in the free -SH content in total protein. This result indicated that more unbonded -SH was involved in gluten protein cross-linking. However, in noodle doughs of Jimai 22 and Ningchun 4, extremely high vacuum degree could go against free thiol oxidation and formation of disulfide bonds in protein, leading to the increased -SH content. This could help explain the observed change in GMP quantity. Vacuum mixing significantly influenced the particle size distributions of GMP, and the appropriate level of vacuum could increase the GMP size. The percentages of volume, surface area and number of large-size GMP with diameter >100 μm in Jimai 22 and Ningchun 4 doughs mixed at 0.06 MPa were significantly higher than at 0 and 0.08 MPa (P<0.05), while the volume percentage of large-size GMP in Zhengmai 366 dough was significantly increased when mixed at 0.08 MPa. The certain vacuum degree could induce a reduction in the free -SH content in GMP for Jimai 22 and Ningchun 4; but for Zhengmai 366, the free -SH content in GMP was not affected by vacuum degree. The results also showed that the difference in GMP content between vacuum mixed dough under 0.06 MPa and non-vacuum mixed dough was significant for Jimai 22 (P<0.05); but for Ningchun 4 and Zhengmai 366, the difference was not significant. Furthermore, we found that the size distribution of GMP in Zhengmai 366 dough was less influenced by the vacuum degree compared with the other 2wheat flours. The phenomenon suggested that the polymeric protein size distribution in noodle dough from weak-gluten flour was more susceptible to the degree of vacuum. In conclusion, the potential gluten protein repolymerization in noodle dough is induced by appropriate vacuum mixing, and the effect of vacuum degree on GMP properties for strong-gluten wheat flour is significantly less than that for weak-gluten flour.