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Background: Tissue flossing bands are a relatively new treatment modality that have increased in popularity in recent years. There is evidence suggesting that tissue flossing could decrease pain and increase joint range of motion, but there is still a need for further studies in this matter. Thus, this study aimed to assess the effect of a single tissue flossing treatment on the perceived pain and upper extremity function among healthy collegiate baseball pitchers. Methods: Twenty apparently healthy collegiate baseball pitchers (age: 21 ± 2.49) participated in this study. Tissue flossing treatment was performed from the wrist to the elbow on the participant’s throwing arm. Before and after treatment, perceived pain and upper extremity function was evaluated. Results: Following one round of tissue flossing treatment, the participants experienced a 27% decrease in perceived pain from baseline (3.05 ± 1.95 to 2.22 ± 2.04, p < 0.05). Participants did not experience a significant increase in perceived upper extremity function. In addition, participants did not report any complications as a result of the intervention. Conclusion: These findings demonstrated that in healthy collegiate baseball pitchers, tissue flossing bands treatment were capable of decreasing perceived pain.