The effect of the order of system assembly was studied in two-stage aqueous two-phase systems loaded with whole bovine blood and BSA. Recovery in the bottom phase of the back extraction varied by up to 40% depending on the manner in which the forward extraction was assembled, significantly impacting upon the efficiency of the process. The effect of mixing intensity was investigated using a simple shear device. As expected, higher shear rates were found to facilitate mass transfer and shorten the time taken to reach equilibrium. Mixing at lower shear rates for extended periods resulted in a lower recovery of protein.