Dough mixing is a key step in the wheat gluten-starch separation process promoting gluten agglomeration. The impact of the water/flour ratio (W/F), mixing speed (N), and wheat cultivar (Orvantis, Caphom, Isengrain) on the protein phase distribution during mixing was studied by macroscopic image analysis. During dough mixing gluten agglomerates grew steadily and finally turned into a filamentous network at optimal dough development (t(peak)). Prior to t(peak), neither W/F nor N impacted the average gluten lump diameter at a fixed stage of mixing. For Orvantis significantly larger gluten agglomerates (up to 272 mu M) were observed as compared to Caphorn and Isengrain (up to 222 and 144 mu M, respectively). Wheat flour cultivar was shown to have an important impact on gluten lump diameter, while mixing parameters (N and W/F) have no direct effect. Mixing parameters merely modulate the absolute gluten lump growth rate, just as they impact the optimal dough development time. (C) 2015 Elsevier Ltd. All rights reserved.