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Background: Atrophy of the rotator cuff is a negative prognostic indicator after rotator cuff repair. Although full-thickness rotator cuff tears accompanied by tendon retraction are commonly associated with decreased muscle cross-sectional area (CSA) on magnetic resonance imaging (MRI), it is unclear whether this is accompanied by histologic atrophy of rotator cuff myofibers. Purpose: To evaluate the effect of supraspinatus tendon retraction and myofiber size on supraspinatus atrophy on MRI. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Supraspinatus muscle biopsy specimens were obtained from consecutive patients undergoing arthroscopic shoulder surgery. Rotator cuff tears were classified according to size. Preoperative MRI was used to measure tendon retraction and CSA of the supraspinatus muscle in the Y-shaped view. The occupation ratio of the supraspinatus was calculated by dividing the supraspinatus CSA by the supraspinatus fossa CSA. Muscle biopsy specimens were examined using laminin to quantify myofiber CSA. The association between supraspinatus tear size and measures of histologic and MRI muscle atrophy were compared using standard statistical tests. Multivariable logistic regression analysis was used to identify independent predictors of muscle atrophy on MRI. Results: A total of 38 patients were included: 8 with no tear, 14 with a partial-thickness tear, and 16 with a full-thickness tear. Increasing tear size was associated with greater distance of tendon retraction (P <.001), smaller mean histologic myofiber size (P =.004), lower mean supraspinatus CSA on MRI (P <.001), and lower occupation ratio: 0.73 (control), 0.66 (partial tear), 0.53 (small to medium full-thickness tear), and 0.38 (large to massive full-thickness) (P <.001). On Pearson correlation analysis, tendon retraction demonstrated strong correlation with occupation ratio (–0.725; P <.001) and weak correlation with myofiber size (–0.437; P =.006), while occupation ratio showed moderate correlation with myofiber size (0.593; P <.001). Multivariable linear regression analysis demonstrated that increasing tendon retraction (P <.001), age (P =.034), and smaller histologic myofiber CSA (P =.047) were independently associated with greater supraspinatus atrophy on MRI. Conclusion: Supraspinatus muscle atrophy appreciated on MRI is independently associated with patient age, tendon retraction, and atrophy of the supraspinatus myofibers at the histologic level. [ABSTRACT FROM AUTHOR]