Abstract OBJECTIVES Transcatheter heart valves (THVs) are investigated according to International Organization for Standardization requirements using in vitro heart simulators to evaluate hydrodynamic performance. In contrast to surgical valves, a THV’s performance heavily depends on the configuration and shape of the aortic anulus. In International Organization for Standardization regulations, there is no detailed definition for the construction of a compartment in which a THV has to be tested. Therefore, the aim of this in vitro study was to compare different in vitro models for functional testing of THVs. METHODS Porcine aortic conduits (23-mm diameter) were implanted in Dacron prostheses and calcified with double-distilled water and calcification buffer at 37°C over 83 million cycles in a Hi-Cycler (durability testing) mimicking nearly 3 patient-years. Hydrodynamic testing of Evolut PRO 26 mm was performed within 3 models (plexiglass, native conduit and calcified conduit; all 23-mm diameter) at a frequency of 64 bpm and different stroke volumes (55–105 ml). RESULTS Calcified conduits showed significantly higher mean pressure gradients (MPG) and lower effective orifice areas (EOA) in comparison to native conduits (without THV; P < 0.001). EOA and MPG of Evolut PRO differed depending on the model tested. Calcified conduits resulted in the lowest EOA and highest MPG of the THV compared to plexiglass and the native conduit. Full expansion of the THV was least impaired in the native conduit, while lowest geometric orifice area, lowest minimal internal diameter and highest pin-wheeling index of Evolut PRO were seen in the calcified conduit. CONCLUSIONS Full expansion and functional performance of the Evolut PRO THV depends on the configuration of the testing compartment in an in vitro setting.