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Introduction: Muscle fatigue is a leading cause of rotator cuff (RC) pathologies. Scapular orientation affected by changes in the thoracic spine account for differences in body postures leading to altered RC muscle activation. This posture-related alteration in RC muscle activation and its fatigue response needs to be analyzed., Materials and Methods: This study included 50 healthy shoulders with no coexisting spine pathologies. Raw data were recorded using electromyography sensors for RC muscles during two isometric maneuvers of abduction and external rotation, performed at 30% maximum voluntary contraction at 30°, 45°, and 90° arm elevation in sitting and standing. The raw data were analyzed in DataLITE ® software, and the mean power frequency (MPF) was extracted to analyze the fatigue response of RC muscles. The Wilcoxon signed-rank test and Kruskal-Wallis test with Bonferroni corrections analyzed fatigue differences between postures and various activities. P < 0.05 was considered significant for the results., Results: Supraspinatus muscle demonstrated significant fatigue at 90° of arm elevation in standing as compared to sitting (MPF -5.40: -5.41; P = 0.03) posture. Between the three elevation angles, all the RC muscles showed increased fatigue at 90° (MPF range -5.22 to -6.64). When compared between abduction and external rotation, only infraspinatus showed fatigue in external rotation (MPF range -5.42 to -6.08). Among all the three RC muscles, infraspinatus showed the maximum fatigue of MPF -6.64 when compared to supraspinatus -5.22 and teres minor -5.36., Conclusion: The findings indicate that alterations in the body postures and different elevation angles affect the RC muscles' fatigue response., (© 2022. The Author(s).)