ABSTRACTThe application of the cold‐ethanol laboratory fractionation method to the bulk separation of wheat starch and gluten is accompanied by incidental dissolution, removal, or redeposition of a small part of the functional gliadin protein. The new distribution resulting from process incidental redeposition of soluble components or by purposeful add‐back of soluble and leached components can lead to differences in functionality and more difficult recovery of native properties. To assess this issue, we exposed several wheat flour types to ethanol and water (50–90% v/v) solutions, water, and absolute ethanol at 22°C and –12°C. The exposure was mass conserving (leached components returned to substrate by evaporation of the solvent without separation of phases) or mass depleting (leached components not returned to substrate). The result of the mass‐conserving contact would be flour with altered protein distributions and intermolecular interactions. The result of the mass‐depleting contact would also include altered protein content. Furthermore, the mass‐conserving contact would model an industrial outcome for a cold‐ethanol process in which leached components would be added back from an alcohol solution. The leaching result was monitored by mixography of the flour, nitrogen analysis, and capillary zone electrophoresis of extracts. Although dough rheology was generally like that of the source flour, there were notable differences. The primary change for mass‐conserving contact was an increase in the time to peak resistance and a decrease in the rate of loss of dough resistance following peak resistance. These changes were in direct proportion to the amount of protein mobilized by the solvent. Leaching at 22°C, prevented dough formation for most aqueous ethanol concentrations and greatly reduced gliadin protein content. Minimal changes were noted for solvent contact at –12°C regardless of the ethanol concentration. The data suggested that 1) the conditions applied in cold‐ethanol enrichment of protein from wheat will generally preserve vital wheat gluten functionality, 2) functionality losses can be recovered by returning the solubilized fractions, and 3) the flour to which the gluten is added may require more mixing.