Background <p><i>Staphylococcus aureus</i> is a major cause of relapsing infection. However, how its phenotypic traits change over time within individual hosts during relapsing infection remains incompletely characterized.</p> Methods <p>In this study, we analyzed five longitudinal <i>S. aureus</i> isolates obtained from two patients with relapsing infection to investigate within-host phenotypic and genomic changes. Whole-genome sequencing was combined with a series of phenotypic assays, including antimicrobial susceptibility testing, erythrocyte lysis assays, whole-blood survival assays, neutrophil extracellular trap (NET) degradation analysis, growth curve determination, antibiotic time–kill assays with levofloxacin, and macrophage phagocytosis and intracellular survival in iBMDMs and murine bloodstream infection models.</p> Results <p>Isolates recovered from each patient were clonally related (ST630/t377 and ST239/t030). Across both clinical trajectories, later isolates showed reduced in vitro growth. Virulence-associated readouts changed over time, including reduced erythrocyte lysis and lower mouse mortality for later isolates in one patient, whereas changes were not uniform between patients. Later isolates displayed improved survival in whole blood, and sequential isolates from one patient showed qualitatively increased NET-degrading activity. Antimicrobial susceptibility profiles remained largely stable, but time–kill assays indicated incomplete eradication under levofloxacin exposure, and later isolates exhibited reduced macrophage phagocytosis and increased intracellular survival.</p> Conclusion <p>Our findings suggest that <i>S. aureus</i> isolates recovered sequentially from individual patients may undergo coordinated, time-associated phenotypic changes during relapsing infection. These observations provide insight into time-associated phenotypic variation of <i>S. aureus</i> during relapsing infection within individual hosts.</p>