Mixing of commercial durum wheat semolina with water was performed under different conditions in a Brabender micromixer equipped with pastamaking shafts. Semolina filling of the mixing chamber was 30.4–42.9% (v/v), shaft speed was 10–110 rpm, temperature was 10–40°C, and hydration level was 47–52.5% (db). The blend of water and semolina evolved from individualized hydrated particles (HP) to a dough product (DP) as a function of these conditions. Torque values (T) and the specific mechanical energies (SME) were recorded during mixing as a function of time. Terms from these curves were defined to characterize the mixing process: to (starting time of dough development), td (time to reach the maximum dough consistency), Tm (mean torque value after dough development), and SMEf (total energy applied to the dough during mixing). Transformation of HP into DP and the mixing temperature were the main parameters affecting to, td, Tm, and SMEf. Protein aggregate distribution was measured by size-exclusion HPLC, protein solubility in 0.01N acetic acid, free -SH content, soluble arabinoxylans, reducing sugars, ferulic acid, carotenoid content, and oxidase activities to characterize the biochemical changes that occurred during pasta dough formation. DP was characterized by lower amounts of insoluble glutenin aggregates, lower protein solubility in dilute acetic acid, lower free -SH content, ferulic acid, carotenoid content, and lower oxidoreductase activities as compared to HP. Once the dough was developed, the effects of mixing speed, temperature, or hydration level on the biochemical composition of the blend were null or low compared to the modifications that were observed when the blends changed from HP to DP. The to and SMEf were the most significant parameters in characterizing the pasta dough mixing process in relation to biochemical changes.