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The effects of in situfreezing and the combination of in situfreezing and stress‐shielding on the microstructure and ultrastructure of the patellar tendon were studied with use of 20 mature rabbits. The patellar tendon was frozen in situwith liquid nitrogen to kill fibroblasts and then was completely released from stress by chronically pulling a stainless‐steel wire installed between the patella and the tibial tubercle, Microstructurally, the freezing treatment induced separation of collagen fiber bundles and fibroblast necrosis at 3 weeks, although the separation disappeared at 6 weeks. Ultrastructurally, small collagen fibrils with a diameter of less than 90 nm were predominant; at 6 weeks, the area occupied by collagen fibrils had decreased. In the frozen‐shielded tendon, numerous large spaces were observed in the matrix at 3 weeks. This treatment increased the number of fibrils with a diameter greater than 360 nm and decreased the number of collagen fibrils per unit of area and the area occupied by collagen fibrils at 3 weeks. This study demonstrated that in situfreezing and the combination of in situfreezing and stress‐shielding leads to a smaller volume of collagen fibrils per unit of cross section of the patellar tendon by mechanisms that remain to be defined.