The present study displays a comprehensive investigation into the micro- and macrostructures of gluten and its responses to temperature-induced changes, employing various analytical techniques. The integration of time domain-nuclear magnetic resonance (TD-NMR), differential scanning calorimetry (DSC), size-exclusion high-performance liquid chromatography (SE-HPLC), field emission scanning electron microscopy (FESEM), solid-state nuclear magnetic resonance (ssNMR), and multiphoton laser microscopy (MLM) measurements facilitates the multidimensional examination of gluten’s phase distribution and structure across various scales. Notably, TD-NMR helps to refine prior T2 assignments for hydrated gluten through dynamic T2 measurements at sub-zero temperatures. The innovative application of TD-NMR uncovers insights into freezable water quantities and their changes under varying temperature conditions. Through real-time analyses utilizing not only TD-NMR but also MLM techniques, along with SE-HPLC measurements, the study highlights increased lacunarities in the gluten structure, particularly between 60 and 85 °C. These structural changes are attributed to heating effects that unfold and denature proteins and culminate in aggregation and crosslinking phenomena, leading to the release of water into macropores, hence changes in the water distribution in the gluten matrix.