In contrast to pellicles formed in vivo, pellicles formed in vitro provide little to no erosion protection for enamel, possibly due to protein degradation from proteases during pellicle formation. With the objective to achieve a more similar effect as observed for in vivo pellicles, the effects of adding protease inhibitors (PI) to saliva in vitro, and/or exchanging saliva repeatedly during pellicle formation were investigated in a cyclic model of pellicle formation and erosion with human enamel specimens. We repeatedly assessed surface microhardness (SMH), measured initial and final surface reflection intensity (SRI), and determined calcium released during erosion. For all the parameters tested, we observed a clear positive effect on erosion protection when adding PI to saliva for pellicle formation: SMH remained harder, SRI remained higher, and less calcium was released. Additionally, exchanging saliva with fresh one during pellicle formation led to a protective effect, but not as strong as the addition of PI. We conclude that adding protease inhibitors to saliva in vitro for pellicle formation leads to an erosion protective effect, which was further increased by repeatedly exchanging the saliva. Whether the pellicle itself more closely resembles in vivo pellicles remains to be investigated.