We present combined experimental and modelling evidence that β-lactoglobulin proteins employed as stabilizers of oil/water emulsions undergo minor but significant conformational changes during premix membrane emulsification processes. Circular Dichroism spectroscopy and Molecular Dynamics simulations reveal that the native protein structure is preserved as a metastable state after adsorption at stress-free oil/water interfaces. However, the shear stress applied to the oil droplets during their fragmentation in narrow membrane pores causes a transition into a more stable, partially unfolded interfacial state. The protein's β-sheet content is reduced by up to 8% in a way that is largely independent of the pressure applied during emulsification, and is driven by an increase of contacts between the oil and hydrophobic residues at the expense of structural order within the protein core.