The protein vital gluten is mainly used for food while interest for non-food applications, like biodegradable materials, increases. In general, the structure and functionality of proteins is highly dependent on thermal treatments during production or modification. This study presents conformational changes and corresponding rheological effects of vital wheat gluten depending on temperature. Dry samples analyzed by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR) and thermalgravimetric analysis coupled with mass spectrometry (TGA-MS) show surface compositions and conformational changes from 25 to 250 °C. Above 170 °C, XPS reveals a decreased N content at the surface while FTIR band characteristics for β-sheets prove structural changes. At 250 °C, protein denaturation accompanied by a significant mass loss due to dehydration and decarbonylation reactions is observed. Oscillatory measurements of optimally hydrated vital gluten describing network properties of the material show two structural changes along a temperature ramp from 25 to 90 °C: at 56–64 °C, the temperature necessary to trigger structural changes increases with the ratio of gliadin to total protein mass, determined by reversed-phase high performance liquid chromatography (RP-HPLC). ... mehrAt a temperature of 79–81 °C, complete protein denaturation occurs. FTIR confirms the denaturation process by showing band shifts with both temperature steps.