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BACKGROUND: Although muscle protein turnover rates have been well established, little is known regarding the turnover of other musculoskeletal tissues. Furthermore, their response to protein supplementation has not yet been assessed. OBJECTIVES: To assess skeletal muscle, bone, cartilage, tendon, ligament, meniscus, and periarticular soft tissue protein synthesis rates over a 2-wk period with or without additional protein supplementation in vivo in older adults. METHODS: Twenty-four otherwise healthy older adults (12M/12F; age: 68 ± 7 y; body mass index: 29 ± 5 kg/m) undergoing total knee arthroplasty participated in this exploratory randomized controlled trial. Participants were randomly allocated to maintain their habitual diet with (n = 12) or without (n = 12) an additional daily bolus of 40 g whey protein for 14 d prior to surgery. Deuterium oxide (HO) was administered throughout the 14-d preoperative period, and all musculoskeletal tissues were collected during subsequent surgery to assess tissue protein synthesis rates. Data are presented as means ± standard deviation. RESULTS: Daily fractional protein synthesis rates (FSRs) in muscle, synovium, and Hoffa's fat pad averaged 1.17% ± 0.23%, 0.79% ± 0.46%, and 0.53% ± 0.29% per day, respectively. Fibrous tissues, including anterior and posterior cruciate ligament, patellar tendon, lateral and medial meniscus, and femoral cartilage FSRs averaged 0.45% ± 0.27%, 0.46% ± 0.32%, 0.20% ± 0.10%, 0.21% ± 0.15%, 0.18% ± 0.14%, and 0.19% ± 0.11% per day, respectively. Finally, FSRs of bone obtained from the distal femur, notch, trochlea, tibia, and patella were 0.18% ± 0.13%, 0.21% ± 0.14%, 0.13% ± 0.10%, 0.12% ± 0.05%, and 0.16% ± 0.10% per day, respectively. FSR differed significantly between tissue groups (P-tissue group < 0.001), with the highest FSR in muscle, followed by periarticular soft tissues, fibrous tissues, and bone (pairwise comparisons, all P < 0.001). Musculoskeletal tissue FSR did not differ between the control and protein supplemented group (P-treatment = 0.27, P-treatment∗tissue type = 0.14). CONCLUSIONS: Muscle, bone, cartilage, tendon, ligament, meniscus, and periarticular soft tissue protein synthesis rates range between 0.12% and 1.17% per day in vivo in older adults. Two weeks of protein supplementation does not seem to increase daily musculoskeletal tissue turnover. This trial was registered at www. CLINICALTRIALS: gov as NCT03037294.