<p>When measured in vitro, the release of metal ions from orthodontic alloys is typically carried out in artificial saliva (AS), a medium with many advantages but lacking the biological complexity of natural human saliva. In this study, we measured ion release profiles from the complete orthodontic fixed appliance, comprising stainless steel and NiTi parts, in a proteinaceous media (yeast extract peptone dextrose, YPD) and compared it to AS. Two immersion models were used, differing in medium replenishment dynamics. To elucidate the metal release results, surface chemistry and topography were analysed using atomic force microscopy (AFM) followed by roughness analysis, and elemental analysis of the top micrometric and nanometric layer (SEM/EDX and XPS analyses).The results showed that proteinaceous media promoted the leaching of Fe, Cu, and Al while suppressing Ni and Cr. Ni²⁺ and Cr³⁺ ions were detected in the top layer on NiTi in AS, but not in YPD. A rough “wavy” surface layer was formed in AS, as opposed to smaller sharper entities formed in YPD. Cu(I) compounds on orthodontic bands were detected in both media. The replenishment of the media during immersion influenced the development of surface chemistry and ion leaching for both types of media, AS and YPD. The results obtained in this study are expected to provide a significant advancement over previous studies using artificial saliva (only).</p> Graphical Abstract <p></p>