Small-angle neutron scattering (SANS) analysis using the TeubnerStrey model has been employed to evaluate the effect of protein incorporation into the middle, bicontinuous microemulsion (B mu E) phase of Winsor-III (W-III) systems formed by an aerosol-OT (AOT)/alkyl ethoxylate mixed surfactant system to understand better the extraction of proteins into and out of B mu Es and to study the effect of proteins on a system that serves as a biomimetic analog of cell membranes. Under conditions of high salinity, the incorporation of positively charged proteins cytochrome c, lysozyme, and a-chymotrypsin, near their solubilization limit in the B mu Es promoted the release of water and oil from the B mu Es, a decrease in the quasi-periodic repeat distance (d), an increase in ordering (a decrease in the amphiphilicity factor, f(a)) for the surfactant monolayers, and a decrease in the surface area per surfactant headgroup, suggesting that the proteins affected the self-assembly of components in the B mu E phase and produced Debye shielding of AOTs sulfonate headgroup. For WIII systems possessing lower salinity, cytochrome c reduced the efficiency of surfactant in the B mu E phase, noted by increases in d and f(a), suggesting that the enzyme and AOT underwent ion pairing. The results of this study demonstrate the importance of ionic strength to modulate proteinsurfactant interactions, which in turn will control the release of proteins encapsulated in the B mu Es, relevant to WIII-based protein extraction and controlled release from B mu E delivery systems, and demonstrate the utility of B mu Es as a model system to understand the effect of proteins on biomembranes.