Background Osteochondral autografts and allografts require mechanical force for proper graft placement into the defect site; however, impaction compromises the tissue. This study aimed to determine the effect of impaction force and number of hits to seat the graft on cartilage integrity. Hypothesis Under constant impulse conditions, higher impaction load magnitudes are more detrimental to cell viability, matrix integrity, and collagen network organization and will result in proteoglycan loss and nitric oxide release. Study Design Controlled laboratory study. Methods Osteochondral explants, harvested from fresh bovine trochleae, were exposed to a series of consistent impact loads delivered by a pneumatically driven device. Each plug received the same overall impulse of 7 Ns, reflecting the mean of 23 clinically inserted plugs. Impaction loads of 37.5 N, 75 N, 150 N, and 300 N were matched with 74, 37, 21, and 11 hits, respectively. After impaction, the plugs were harvested, and cartilage was analyzed for cell viability, histology by safranin-O and picrosirius red staining, and release of sulfated glycosaminoglycans (GAGs) and nitric oxide. Data were compared with nonimpacted controls. Results Impacted plugs had significantly lower cell viability than nonimpacted plugs. A dose-response relationship in loss of cell viability with respect to load magnitude was seen immediately and after 4 days but lost after 8 days. Histological analysis revealed intact cartilage surface in all samples (loaded or control), with loaded samples showing alterations in birefringence. While the sulfated GAG release was similar across varying impaction loads, release of nitric oxide increased with increasing impaction magnitudes and time. Conclusion Impaction loading parameters have a direct effect on the time course of the viability of the cartilage in the graft tissue. Clinical Relevance Optimal loading parameters for surgical impaction of osteochondral grafts are those with lower load magnitudes and a greater number of hits to ensure proper fit.