It is presumed that structural and functional alterations of biopolymers, which occur during grinding, are caused by a mechanical modification of polymers. As a result, thermally induced changes of flours are neglected. In this study, the impact of thermo-mechanical stress (TMS), as occurring during general grinding procedures, was further differentiated into thermal stress (TS) and mechanical stress (MS). For TS, native wheat flour, as well as the purified polymers of wheat—starch and gluten—were heated without water addition up to 110 ∘ C. Isolated MS was applied in a temperature-controlled ultra-centrifugal grinder (UCG), whereby thermal and mechanical treatment (TMS) was simultaneously performed in a non-cooled UCG. TS starch (110 ∘ C) and reference starch did not show differences in starch modification degree (2.53 ± 0.24 g/100 g and 2.73 ± 0.15 g/100 g, AACC 76-31), gelatinization onset (52.44 ± 0.14 ∘ C and 52.73 ± 0.27 ∘ C, differential scanning calorimetry (DSC)) and hydration properties (68.9 ± 0.8% dm and 75.8 ± 3.0%, AACC 56-11), respectively. However, TS led to an elevated gelatinization onset and a rise of water absorption of flours (Z-kneader) affecting the processing of cereal-based dough. No differences were visible between MS and TMS up to 18,000 rpm regarding hydration properties (65.0 ± 2.0% dm and 66.5 ± 0.3% dm, respectively). Consequently, mechanical forces are the main factor controlling the structural modification and functional properties of flours during grinding.