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Abstract In-vitro testing is widely used to evaluate the hemodynamic performance of transcatheter aortic valves (TAVs). However, many studies assess self-expanding valves using blood analogs matched in viscosity at room temperature, despite the temperature-dependent mechanical behavior of nitinol stent frames. This study investigates the independent and combined effects of temperature and viscosity on the in-vitro performance assessment of a self-expanding TAV. Rheological characterization was performed to identify water-glycerin solutions with matched viscosities at room temperature and physiological temperature (37°C). A patient-specific aortic model was cast in silicone. A self-expanding Medtronic Evolut R valve was deployed within the compliant model and evaluated using a pulse-duplicating left heart simulator under physiological flow and pressure conditions. The transvalvular pressure gradient and effective orifice area (EOA) were calculated. Rheometry identified two fluid conditions with comparable viscosities at room and body temperatures. Heating the blood analog to 37°C resulted in significantly reduced transvalvular pressure gradient and increased EOA compared to the viscosity-matched room temperature condition (p<0.0001). At physiological body temperature, the higher viscosity fluid (42% glycerin) yielded further improvements in valve performance relative to the lower viscosity fluid(38% glycerin), with lower pressure drop and larger EOA (p<0.0001). These results demonstrate that temperature dependent effects influence the hemodynamic performance of self-expanding nitinol TAVs beyond viscosity matching alone. Combined consideration of temperature and viscosity is therefore necessary to ensure physiologically relevant in-vitro assessment of self-expanding TAVs.