The adipose tissue-derived stromal vascular fraction (SVF) is a promising candidate for use in cell therapy and tissue engineering due to its regenerative and immunomodulatory properties. Some therapies are based on using the complete SVF product, whereas others depend on the expansion of adipose-derived stromal cells (ASCs) in culture. The latter application often involves a time delay between adipose tissue harvest and SVF isolation. This study investigated how storage time and temperature affected cell quality and composition. Aliquots of lipoaspirate were stored cold (4°C), at room temperature (18-20°C), or at 37°C. SVF was isolated on sequential time points over a period of 48 h, and the following were assessed: Cell viability, vitality, composition, and the proliferative potential of the ASCs. When the lipoaspirate was stored cold, the viability of the SVF remained stable for up to 48 h; however, the vitality of the SVF decreased significantly after 24 h. When stored at higher temperatures (room temperature or 37°C), the vitality of the SVF decreased after 8 h. The ASC fraction in the SVF decreased rapidly after 8 h when stored at higher temperatures, whereas this change was delayed significantly when the lipoaspirate was stored cold. Tendencies towards increases in the lag phase, population doubling time (PDt), and time to reach confluency were observed when the lipoaspirate was stored at higher temperatures. The vitality of the SVF was correlated significantly with the time of the lag phase and the time required to reach confluence, whereas no correlation was observed with the PDt. Both prolonged storage time and increased temperature during lipoaspirate storage negatively affected the quality of the obtained SVF. Our results suggest that lipoaspirate should be stored for no longer than 24 h at 4°C to maintain the optimal quality for the isolation of SVF and the expansion of ASCs.