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Thus, the purpose of this study was to understand the timeline of protease activity in the soft tissues of the shoulder (tendon, muscle, and cartilage) that may lead to down\u2010stream degeneration following rotator cuff tear. A well\u2010established rat model involving suprascapular nerve denervation and supraspinatus\/infraspinatus tendon transection was employed. Histological staining and\/or micro\u2010computed tomography (\u00b5CT) were used to observe structural damage in the supraspinatus tendon and muscle, humeral head cartilage, and subchondral bone. Multiplex gelatin zymography was utilized to assess protease activity in the supraspinatus tendon and muscle, and humeral head cartilage. Zymography analysis demonstrated that cathepsins were upregulated in the first week in all tissues, while MMP\u20102 maintained prolonged activity in supraspinatus tendon between 1 and 3 weeks and increased only at 3 weeks in supraspinatus muscle. In supraspinatus tendon, increased cathepsin L and MMP\u20102 activity in the first week was concurrent with matrix disorganization and infiltration of inflammatory cells. In contrast, significant upregulation of cathepsin L and K activity in supraspinatus muscle and humeral head cartilage did not correspond to any visible tissue damage at 1 week. However, focal defects developed in half of all animals\u2019 humeral head cartilage by 12 weeks (volume: 0.12\u2009\u00b1\u20090.09\u2009mm<jats:sup>3<\/jats:sup>). This work provides a more comprehensive understanding of biochemical changes to joint tissue over time following rotator cuff tear. Overall, this provides insight into potential therapeutic targets and will better inform ideal intervention times and treatments for each tissue. \u00a9 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 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