Background <p>Extracellular vesicles (EVs) are emerging as non-invasive biomarkers in cancer, but their role in monitoring the response to neoadjuvant systemic treatment (NST) in patients with breast cancer remains unclear. This study aimed to assess whether EV concentration and surface marker profiles in plasma reflect treatment response and clinical outcome in patients with early-stage breast cancer receiving NST.</p> Methods <p>Plasma samples were collected from 59 patients with luminal B-like, HER2-positive, or triple-negative breast cancer before and after NST. EVs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy, Western blotting, and MACSPlex surface marker profiling. Paired samples were available for 29 patients, allowing longitudinal analysis.</p> Results <p>Patients who achieved pathological complete response (pCR) had significantly lower baseline EV concentrations than those with residual disease. Post-treatment EV levels were also lower in patients who remained free from distant metastasis and had improved breast cancer-specific survival. EV surface marker profiling revealed that CD69, CD29, and CD49e were reduced after NST, whereas CD44 was increased. Notably, EpCAM levels increased specifically in non-PCR patients, suggesting persistent tumor-derived EV release, whereas SSEA-4 levels increased only in patients who achieved pCR. Although EV concentrations and markers differed by subtype and outcome, changes in EV levels following NST were not independently predictive of prognosis.</p> Conclusions <p>These findings support the potential of plasma-derived EVs as dynamic biomarkers of treatment response, and suggest possible prognostic relevance in breast cancer. Validation in larger, independent cohorts is needed to assess their clinical applicability.</p>