BACKGROUND: Adipose-derived stem cells (ADSCs) and the stromal vascular fraction (SVF) have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions. Traditional enzymatic methods for isolating these cells face challenges such as high costs, lengthy processing time, and regu-latory complexities. AIM: This systematic review aimed to assess the efficacy and practicality of non-enzymatic, mechanical methods for isolating SVF and ADSCs, comparing these to conventional enzymatic approaches. METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a comprehensive literature search was conducted across multiple databases. Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue. The risk of bias was assessed, and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies. RESULTS: Nineteen studies met the inclusion criteria, highlighting various mechanical techniques such as centrifugation, vortexing, and ultrasonic cavitation. The review identified significant variability in cell yield and viability, and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures. Despite some advantages of mechanical methods, including reduced processing time and avoidance of enzymatic reagents, the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation. CONCLUSION: Non-enzymatic, mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs, potentially simplifying the isolation process and reducing regulatory hurdles. However, further research is necessary to standardize these techniques and ensure consistent, high-quality cell yields for clinical applications. The development of efficient, safe, and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.