Background <p>Microvascular fragments (MVFs) are intact vascular segments derived from adipose tissue that possess considerable potential for promoting tissue vascularization in regenerative medicine. However, conventional single-step enzymatic digestion methods often lead to incomplete adipose tissue dissociation and poor MVF quality.</p> Methods <p>We developed and validated a sequential enzymatic digestion protocol optimized for isolating MVFs from human lipoaspirate. Adipose samples were processed using either a conventional one-step collagenase digestion or a three-step sequential method. MVFs were evaluated for yield, viability, structural integrity, cellular phenotype, and angiogenic function both in vitro and in vivo.</p> Results <p>Compared with the conventional approach, the sequential protocol produced a 2.2-fold increase in MVF yield and significantly reduced undigested tissue residues (p &lt; 0.0001). MVFs isolated by the sequential protocol showed superior cell viability (93.3% vs. 75.6%), a greater proportion of long fragments, preserved endothelial and perivascular architecture, and enhanced angiogenic performance in collagen gel assays and mouse subcutaneous implantation models.</p> Conclusions <p>This optimized sequential digestion protocol enables the efficient and producible isolation of high-quality MVFs from human adipose tissue. It holds great promise for applications in vascularized tissue engineering and regenerative therapies.</p> No Level Assigned <p>This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors <a href="http://www.springer.com/00266">www.springer.com/00266</a>.</p> Graphical Abstract <p></p>