Whole-organ engineering-based on the functional repopulation of acellular whole-organ scaffolds derived from perfusion-based in toto decellularization of the specific organ system-is one of the most promising fields in tissue engineering. However, to date, we still have hardly any insights into the process of perfusion-based scaffold generation itself, with human-scale scaffolds usually obtained by adoption of small animal decellularization models, although those organs are of decreased biomass and potentially different biological characteristics. Therefore, in this study we analyzed perfusion-based human-scale whole-heart decellularization by evaluating and comparing the dynamics of biomass discharge and its kinetic characteristics during in toto decellularization of ovine and rodent hearts, while introducing a theoretical model of biomass depletion during perfusion-based whole-heart decellularization. Our results suggest highly varying process characteristics for the in toto decellularization of individual human-scale organs, such as protein discharge kinetics or time-dependent viscoelasticity of formed debris, despite seemingly consistent inter-sample decellularization efficacy, as evaluated by conventional disruptive analysis of obtained ECM scaffolds. Hence, the here exposed insights into the mechanistics of whole-heart decellularization as well as the introduced non-disruptive process accompanying tools may help to monitor and further optimize the decellularization process, especially with regards to human-scale scaffold production.