This single-center prospective observational study evaluated peri-procedural changes in circulating markers of endothelial glycocalyx injury in patients with chronic limb-threatening ischemia undergoing lower extremity endovascular treatment. Fifty patients (mean age 72.2 years; 56% with diabetes mellitus; 42% current smokers) underwent percutaneous transluminal angioplasty, most commonly involving the femoropopliteal segment and using 4F introducer sheaths. Serum concentrations of syndecan-1, heparan sulfate, and hyaluronic acid were measured using ELISA before and one day after the procedure. No significant differences were observed between baseline and post-procedural biomarker levels, with minimal median changes, overlapping interquartile ranges, and negligible paired effect sizes. Although individual biomarker trajectories demonstrated considerable interindividual variability, no consistent directional trend was identified. Furthermore, demographic and clinical factors, including age, sex, diabetes, and smoking status, as well as procedural variables such as stent implantation, sheath size, and intervention level, were not associated with biomarker changes in univariate or multivariable analyses. These findings suggest that contemporary endovascular revascularization is biologically well tolerated and does not induce detectable systemic endothelial glycocalyx disruption. Nevertheless, the interpretation of these findings is limited by the moderate sample size and single-center design of the study. In addition, syndecan-1, heparan sulfate, and hyaluronic acid are not exclusive markers of endothelial glycocalyx injury and may also reflect extracellular matrix turnover and tissue remodeling.