The change in mitral valve morphology and hemodynamic performance following fixation was investigated. Porcine mitral valves were studied in vitro with a rigid left heart simulator under physiological conditions before and after glutaraldehyde fixation, genipin fixation, and cryopreservation, while maintaining cardiac output (CO). Anatomic measurements, hydrodynamic data, video and ultrasound imaging, and three-dimensional papillary muscle (PM) force measurements were used to assess valve performance. The cryopreserved valves did not show any statistically significant morphological or hydrodynamic performance differences after preservation. After glutaraldehyde and genipin fixation, the forward flow opening area, the PM force, the regurgitant volume, and the work required by the heart simulator to maintain a constant CO at constant vascular resistance was altered. Chemical fixation of porcine mitral valves adversely affects the hemodynamics of the valves, increasing overall workload. The effects were more severe after genipin fixation than glutaraldehyde fixation.