The rapid visco analysis (RVA) system was used to measure rheological behaviour in 20% (w/v) gluten-in-water suspensions upon applying temperature profiles. The temperature profiles included a linear temperature increase, a holding step, a cooling step with a linear temperature decrease to 50 °C, and a final holding step at 50 °C. Temperature and duration of the holding phase both affected RVA viscosity and protein extractability. Size-exclusion and reversed-phase HPLC showed that increasing the temperature (up to 95 °C) mainly decreased glutenin extractability. Holding at 95 °C resulted in polymerisation of both gliadin and glutenin. Above 80 °C, the RVA viscosity steadily increased with longer holding times while the gliadin and glutenin extractabilities decreased. Their reduced extractability in 60% ethanol showed that γ-gliadins were more affected after heating than α-gliadins and ω-gliadins. Enrichment of wheat gluten in either gliadin or glutenin showed that both gliadin and glutenin are necessary for the initial viscosity in the RVA profile. The formation of polymers through disulphide bonding caused a viscosity rise in the RVA profile. The amounts of free sulphydryl groups markedly decreased between 70 and 80 °C and when holding the temperature at 95 °C.