Background/Objectives: Although the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age-and BMI-matched) donors must exist.Subjects/Methods: Using Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined.Results: Dissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity.Conclusions: Collectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose. (C) 2018 The Authors. Published by Elsevier GmbH.