Adipose tissue is an abundant source of autologous adult stem cells that may bring new therapeutic perspectives on the treatment of diabetes and its complications. It is unclear whether adipose tissue-derived stromal cells (ASCs) of diabetic patients, constantly influenced by hyperglycaemia, have the same properties as non-diabetic controls. As an alternative source of ASCs, adipose tissue from distal limbs of diabetic patients with critical ischemia was isolated. ASCs were characterized in terms of cell surface markers, multilineage differentiation and the expression of vascular endothelial growth factor (VEGFA), chemokine-related genes and compared with non-diabetic controls. Flow cytometry analysis confirmed mesenchymal phenotypes in both diabetic and non-diabetic ASCs. Nevertheless, 40% of diabetic and 20% of non-diabetic ASC samples displayed high expressions of fibroblast marker, which inversely correlated with the expression of CD105. In diabetic patients, significantly decreased expression of VEGFA and chemokine receptor CXCR4 was found in fibroblast-positive ASCs, compared with their fibroblast-negative counterparts. Reduced osteogenic differentiation and the downregulation of chemokine CXCL12 were found in fibroblast-negative diabetic ASCs. Both diabetic and non-diabetic ASCs were differentiated into adipocytes and chondrocytes and did not reveal islet-like cell differentiation. According to this study, adipose tissue from distal limbs of diabetic patients is not satisfactory as an autologous ASC source. Hyperglycaemic milieu as well as other metabolic disorders linked to diabetes may have an influence on endogenous stem cell properties. The present study investigated the feasibility of autologous stem cell therapy in diabetic patients. ASCs isolated from the ischemic limb of diabetic patients were found to be less potent when compared phenotypically and functionally to control non-diabetic counterparts with no signs of limb ischemia. High expression of fibroblast markers associated with reduced expression of VEGFA as well as reduced osteogenic differentiation may have an impact on the effectiveness of autologous cell therapies in diabetic patients.