Updated publication reference for PubMed record(s): 32393437. Turnover of matrix proteins is essential to maintain tissue homeostasis, and dysregulation of protein turnover has been implicated in a variety of diseases. However, proteome dynamics in the musculoskeletal system, particularly tendon, remain relatively unexplored. The aim of this study was therefore to calculate the turnover rate of individual proteins within tendon and its constituent components. We hypothesised that turnover of proteins in the interfascicular matrix would be greater than in the fascicles. Achilles tendons were harvested from rats fed heavy labelled water over a period of 127 days. Proteins were extracted from one Achilles tendon, while laser capture microdissection was used to isolate regions of interfascicular and fascicular matrix from the other Achilles prior to protein extraction. Samples were analysed using tandem mass spectrometry and the rate of label incorporation used to calculate turnover rate of individual proteins. Results demonstrate complex proteome dynamics within tendon, with differences in protein turnover rates approaching 1000-fold. Collagen turnover was relatively slow, with much more rapid turnover of proteoglycans and glycoproteins. Data support the hypothesis, demonstrating overall faster protein turnover within the interfascicular matrix compared to the fascicles. More rapid turnover of the interfascicular matrix may be a mechanism to repair microdamage to this region which is exposed to high levels of shear during locomotion. The techniques used here provide a powerful approach to interrogate alterations in protein turnover in the musculoskeletal system with ageing and/or disease which are likely to influence injury risk.