Clinical and basic science applications using adipose-derived stem cells (ADSCs) are gaining popularity. The current adipose tissue harvesting procedures introduce nonphysiological conditions, which may affect the overall performance of the isolated ADSCs. In this study, we elucidate the differences between ADSCs isolated from adipose tissues harvested within the first 5 min of the initial surgical incision (well-vascularized, nonpremedicated condition) versus those isolated from adipose tissues subjected to medications and deprived of blood supply during elective free flap procedures (ischemic condition). ADSCs isolated from well-vascularized and ischemic tissues positively immunostained for several standard stem cell markers. Interestingly, the percent change in the CD36 expression for ADSCs isolated from ischemic versus well-vascularized tissue was significantly lower in males than females (p p < 0.05). These results indicate that ADSCs isolated from ischemic tissue either fail to uptake fatty acids or fail to efficiently convert those fatty acids into triglycerides. Therefore, more robust ADSCs suitable to establish in vitro adipose tissue models can be obtained by harvesting well-vascularized and nonpremedicated adipose tissues.