In this work, experiments were performed to define operating windows for processing parameters for the isolation of oil bodies from soybeans in a mild way. An aqueous extraction process for simultaneous separation of oil bodies and proteins from soybean was tested. The effect of the particle size on the extraction of oil bodies with two different grinding methods, to obtain one coarse flour (d90 300 μm) and one fine flour (d90 40 μm) was studied. The extractability of the coarse flour was better as compared to fine flour: oil recoveries from the cream were very similar (23% and 24.5% of the total soybean oil), and the protein extraction yield was higher for the coarse flour (48% against 40% of the total protein). To enhance the extraction yield of the protein and the oil, three different pretreatments were applied before the aqueous extraction process took place. The pretreatments include: enzymatic hydrolysis, ultrasound and the combination of the two. We found that the pretreatment with ultrasound reduced the remaining insoluble fraction and increased the amount of solids extracted into the aqueous phase. The combination of ultrasound and enzymes resulted in the cream with the highest lipid-to-protein ratio of 10:1. Different aqueous extraction process alternatives were compared with a benchmark process (neither enzymes nor ultrasound).