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Optimization of bread dough formulation with defatted wheat germ (DWG) in terms of dough rheological properties was the objective of this study. Fractional factorial design (Formula presented.) was applied in order to investigate influence of DWG granulation (<150 µm and 150−1000 µm), DWG content (10 and 20%), addition of gluten (0 and 5%), ascorbic acid (0.004 and 0.008%) and combined improver (0.1 and 0.3%) on farinograph and extensograph parameters. Responsive variables were water absorption and the degree of softening, obtained by farinograph, and resistance to extension and extensibility as well as their ratio, obtained by extensograph analysis. Gluten addition expressed dominant positive effect on water absorption and the degree of softening. The most pronounced negative effect on dough resistance exhibited DWG content (contribution of 57%). The combination of DWG with smaller particle size and 5% of gluten addition increased both resistance and extensibility, but R/E ratio was not affected. The optimal values for DWG granulation, DWG content, gluten content, ascorbic acid content and combined improver content were: < 150 µm, 14.24%, 4.83%, 0.004% and 0.1%, respectively, while predicted values for desired responsible variables were: water absorption 65.90%, degree of softening 84.05 FU, resistance 301.30 EU, extensibility 108.55 mm and R/E 2.5. © The Author(s) 2022.