In animal models, the physiological systems involved in metabolic homeostasis exhibit a sex difference. Investigators often use male rodents because they show metabolic disease better than females. Thus, females are not used precisely because of an acknowledged sex difference that represents an opportunity to understand novel factors reducing metabolic disease more in one sex than the other. The National Institutes of Health (NIH) mandate to consider sex as a biological variable in preclinical research places new demands on investigators and peer reviewers who often lack expertise in model systems and experimental paradigms used in the study of sex differences. This Perspective discusses experimental design and interpretation in studies addressing the mechanisms of sex differences in metabolic homeostasis and disease, using animal models and cells. We also highlight current limitations in research tools and attitudes that threaten to delay progress in studies of sex differences in basic animal research.