Decellularized aortic valves (AV) are promising scaffolds for tissue engineered heart valve (TEHV) application; however, it is not known what the intrinsic fatigue mechanism of the AV extracellular matrix (ECM) is and how this relates to decellularized AV functional limits when tissue remodeling does not take place. In this study, decellularized AVs were subjected to in vitro cardiac exercising and the exercised leaflets were characterized to assess the structural and mechanical alterations. A flow-loop cardiac exerciser was designed to allow for pulsatile flow conditions while maintaining sterility. The acellular valve conduits were sutured into a silicone root with the Valsalva sinus design and subjected to cardiac cycling for 2 weeks (1.0 million cycles) and 4 weeks (2.0 million cycles). Following exercising, thorough structural and mechanical characterizations were then performed. The overall morphology was maintained and the exercised leaflets were able to coapt and support load; however, the leaflets exhibited an unfolded and thinned morphology. The straightening of the locally wavy collagen fiber structure was confirmed by histology and small angle light scattering; the disruption of elastin network was also observed. Biaxial mechanical testing showed that the leaflet extensibility was largely reduced by cardiac exercising. In the absence of cellular maintenance, decellularized leaflets experience structural fatigue due to lack of exogenous stabilizing crosslinks, and the structural disruption is irreversible and cumulative. Although not being a means to predict the durability of the acellular valve implants, this mechanistic study reveals the fatigue pattern of the acellular leaflets and implies the importance of recellularization in developing a TEHV, in which long term durability will likely be better achieved by continual remodeling and repair of the valvular ECM.