Objective <p>Mesenchymal stem cells (MSCs) hold substantial promise in regenerative medicine owing to their immunomodulatory, neuroregenerative, and self-renewal properties. Adipose tissue (AT) serves as an optimal MSC source due to its high yield and rapid proliferation. This study evaluated the safety and exploratory clinical effects of non-cryopreserved, culture-expanded autologous AT-MSCs in patients with secondary progressive multiple sclerosis (SPMS).</p> Methods <p>High-dose fresh autologous AT-MSCs (4.4 × 10⁶ ± 1.7 × 10⁶ cells) were intravenously administered to 10 female patients with SPMS (Expanded Disability Status Scale [EDSS] score 4–6) in two doses, seven days apart. Adverse events were monitored for 9 months post-transplantation. Magnetic resonance imaging (MRI) assessments quantified lesion number, volume, and contrast-enhancing lesions. EDSS scores, depression, and quality-of-life measures were evaluated over 9 months. MSC immunomodulatory effects were assessed via gene expression of inflammatory and anti-inflammatory cytokines and peripheral blood regulatory T-cell (Treg) proportions.</p> Results <p>No serious adverse events occurred over 9 months. AT-MSC therapy reduced T2-FLAIR lesion number and volume, improved EDSS scores, and enhanced psychological outcomes. It also increased Treg cell proportions and anti-inflammatory cytokine expression while decreasing inflammatory cytokines.</p> Conclusion <p>High-dose fresh AT-MSCs appear safe and well-tolerated in SPMS patients, with promising exploratory clinical benefits. These findings support AT-MSCs as a potential multiple sclerosis therapy.</p> <p><i>Trial registration</i> Registered with the Iranian Registry of Clinical Trials (Reference: IRCT20091127002778N1 at 2018-01-10).</p>