Purpose: Acute exercise redistributes large numbers of memory T cells, which may contribute to enhanced immune surveillance in regular exercisers. It is not known, however, if acute exercise promotes a broad or oligoclonal T-cell receptor (TCR) repertoire or evokes transcriptomic changes in "exercise-responsive" T-cell clones. Methods: Healthy volunteers completed a graded bout of cycling exercise up to 80% V̇O2max. DNA was extracted from peripheral blood mononuclear cells collected at rest, during exercise (EX), and 1 h after (+1H) exercise, and processed for deep TCR-β chain sequencing and tandem single-cell RNA sequencing. Results: The number of unique clones and unique rearrangements was decreased at EX compared with rest (P < 0.01) and +1H (P < 0.01). Productive clonality was increased compared with rest (P < 0.05) and +1H (P < 0.05), whereas Shannon's Index was decreased compared with rest (P < 0.05) and +1H (P < 0.05). The top 10 rearrangements in the repertoire were increased at EX compared with rest (P < 0.05) and +1H (P < 0.05). Cross-referencing TCR-β sequences with a public database (VDJdb) revealed that exercise increased the number of clones specific for the most prevalent motifs, including Epstein–Barr virus, cytomegalovirus, and influenza A. We identified 633 unique exercise-responsive T-cell clones that were mobilized and/or egressed in response to exercise. Among these clones, there was an upregulation in genes related to cell death, cytotoxicity, and activation (P < 0.05). Conclusions: Acute exercise promotes an oligoclonal T-cell repertoire by preferentially mobilizing the most dominant clones, several of which are specific to known viral antigens and display differentially expressed genes indicative of cytotoxicity, activation, and apoptosis. [ABSTRACT FROM AUTHOR]