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Mechanical characterization of the ex vivo tricuspid valve (TV) continues to provide key insights into native valve function and the development of valvular diseases. However, experimental methods to characterize TV biomechanical behavior ex vivo often fail to account for potential changes in the tissue’s mechanical responses that may occur during experiment preparation. Therefore, we assessed the mechanical responses of the anterior tricuspid leaflet (ATL) via biaxial mechanical testing over the course of 5 h to validate the accuracy of our fresh tissue experiments. We hypothesized that ATL mechanical responses would remain consistent for the proposed time scale. We found that ATL stiffness, represented by the upper tangent modulus (UTM), did not significantly change in either the radial or circumferential directions for the 5-h test period. Similarly, no significant change was observed in radial or circumferential strains corresponding to an estimated mean systolic stress value of 85 kPa. Overall mean UTM (±standard error of the mean (SEM)) showed that ATL samples were significantly stiffer in the circumferential direction (11.3 ± 0.98 MPa) compared to the radial direction (2.29 ± 0.20 MPa) across all time points. Thus, our results indicate that the outcomes of ex vivo tricuspid valve studies requiring sample preparation up to 5 h remain reliable.