High molecular weight glutenin subunits (HMW-GS) were isolated from wheat flour and polymerized in vitro at pH 3.0 with different oxidizing agents (KBrO3, KIO3, H2O2). An oxidation protocol with single addition of oxidant (single-step oxidation) was compared with a set-up in which the oxidant was added in multiple steps (stepwise oxidation). Changes in size distribution were evaluated with size-exclusion HPLC, multilayer SDS-PAGE, and flow-field flow fractionation (flow-FFF). Flow-FFF is particularly suitable for measuring changes in glutenin size in the very high size ranges. In order of increasing sizes of the resulting polymers, the different oxidizing agents could be ranked as KBrO3 < KIO3 < H2O2. However, none of the oxidation conditions allowed for a complete polymerization of HMW-GS. Interestingly, it was found that high concentrations of KIO3 negatively affect the degree of polymerization. A similar observation was not made with KBrO3 or H2O2 SDS-PAGE showed that y-type HMW-GS particularly failed to incorporate in glutenin polymers. Simultaneously, these HMW-GS displayed higher mobilities on SDS-PAGE that can be ascribed to the formation of intrachain SS bonds. Possible explanations for the incomplete polymerization of HMW-GS are given.