To test the effects of load duration on the joint changes, we subjected the left tibiae of twenty-one young (10-week-old) C57Bl/6 male mice (Jackson Laboratories, Bar Harbor, ME) to cyclic compressive loading for 1, 2, and 6 weeks at a 9.0N peak load. We also loaded the tibiae of forty-two adult (26-week-old) C57Bl/6 male mice at 4.5N and 9.0N peak loads for 1, 2, and 6 weeks. A load level of 9.0N generates 1200με in the tibial mid-shaft of 10-week-old mice based on in vivo strain gauging (20 (link)). In vivo tibial loading was applied for 1200 cycles at 4 Hz for 5 days per week at each peak load under general anesthesia (2% Isoflurane, 1.0 L/min, Webster). The applied loading was based on protocols demonstrated previously to have an anabolic effect on the tibial metaphysis in growing and adult mice (Supp Figure 1) (20 (link)–22 (link)). The left limb was loaded, and the right limb served as the non-loaded control. In preliminary studies in our laboratory, we found that metaphyseal bone mass and architecture of the non-loaded control (right) limbs were not affected by loading of the left limbs. After the specified duration, the mice were euthanized, and the intact knee joints were dissected and fixed in 10% formalin overnight. All experimental procedures were approved by the Institutional Animal Care and Use Committee.