Background <p>A growing number of studies show that physical exercise is feasible and safe for cancer patients and enhances various physical and cognitive capabilities, thus improving the quality of life and disease outcomes. The molecular mechanisms behind these effects are, however, poorly understood. Here, we present the results from the CancerBeat study, where breast cancer (BC) patients receiving neoadjuvant chemotherapy (NAC) were enrolled in a high-intensity interval training (HIIT) program to investigate the effects of exercise on tumor biology and response to NAC.</p> Methods <p>Fifty BC patients were randomized into HIIT and control (CON) groups. Patients were enrolled in the study shortly after diagnosis, and the HIIT group took personalized home-based training sessions for the whole duration of NAC (mean 24 weeks). NAC response was evaluated using the standard Miller-Payne grading system. A retrospective control (Ctrl) group consisted of age, stage and subtype-matched BC patients receiving standard of care during NAC. Acute exercise-induced changes in the plasma proteome were analyzed with the Olink Immuno-Oncology panel. Bulk RNA sequencing analysis was performed on BC and normal breast tissues from 20 patients in the HIIT and Ctrl groups. Tumor-infiltrating immune cell composition was inferred from the bulk RNA-seq data using CIBERSORTx, and findings were validated with a 7-plex immunofluorescence assay.</p> Results <p>In the HIIT group, the proportion of patients with Miller-Payne grades 4 and 5 was significantly higher compared to CON (80% vs. 47%, <i>p</i> = 0.036) and Ctrl groups (80% vs. 50%, <i>p</i> = 0.047), representing the primary finding of this study. Exploratory plasma proteomic analysis identified 2 and 20 plasma proteins whose levels change after acute exercise in BC patients at diagnosis and after completing the training program/NAC, respectively; five of these had not previously been linked to exercise. Bulk RNA sequencing of post-NAC tumor tissues revealed HIIT/NAC-associated gene expression patterns consistent with less aggressive phenotypes, favorable outcomes, and altered wound-healing and immune-related processes at the tumor site. The findings suggest the possible involvement of fibroblasts, endothelial cells, and other stromal cells in exercise-associated tumor microenvironment remodeling. CIBERSORTx analysis suggested greater infiltration of activated NK cells in HIIT tumors compared to resting NK cells in Ctrl tumors. As an exploratory observation, both plasma and tumor tissue levels of decorin were upregulated in the HIIT group, and this increase was associated with better NAC response and favorable prognosis. These findings warrant further investigation in larger, dedicated studies.</p> Conclusions <p>Our study provides novel insights into the molecular mechanisms underlying the protective effects of exercise on BC progression. These preliminary results support the further investigation of physical exercise as a component of BC treatment and highlight the need for clinical trials exploring the impact of HIIT on immunotherapy efficacy.</p> Trial registration <p>retrospectively registered at ClinicalTrials.gov, NCT06522971.</p>