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Background: BVF can be used to improve THV hemodynamics following valve-in-valve (VIV) intervention. However, whether BVF should be performed before or after THV deployment and implications on durability is unknown. Aims: To assess the impact of bioprosthetic valve fracture (BVF) timing on long-term transcatheter heart valve (THV) durability. Methods : The impact of BVF timing was assessed using small ACURATE neo (ACn) or 23mm SAPIEN 3 (S3) THVs deployed in 21mm Mitroflow valves compared to no-BVF controls. Valves underwent accelerated wear testing (AWT) up to 200M cycles (equivalent to 5 years). At 200M cycles, THVs were evaluated by hydrodynamic testing, second harmonic generation (SHG) microscopy, scanning electron microscopy (SEM) and histology. Results: At 200M cycles, the regurgitant fraction (RF) and effective orifice area (EOA) for the ACn were respectively 8.03±0.30%/1.74±0.01cm 2 (no BVF), 12.48±0.70%/1.97±0.02cm 2 (BVF before VIV) and 9.29±0.38%/2.21±0.0cm 2 (BVF after VIV). For the S3 these values were 2.63±0.51%/1.26±0.01cm 2 , 2.03±0.42%/1.65±0.01cm 2 , and 1.62±0.38%/2.22±0.01cm 2 respectively. Further, SHG and SEM revealed a higher degree of superficial leaflet damage when BVF was performed after VIV for ACn and S3. However, histological analysis, revealed significantly less damage, determined by matrix density analysis, through the entire leaflet thickness when BVF was performed after VIV with the S3 and a similar but non-significant trend in ACn. Conclusion: BVF performed after VIV appears to offer superior long-term EOA without increased RF. Ultra-structure leaflet analysis reveals that timing of BVF can differentially impact leaflets, with more superficial damage but greater preservation of overall leaflet structure when BVF is performed after VIV.