Ketogenic diets (KDs) are widely used in preclinical research to investigate metabolism and neurological function, yet many studies lack methodological consistency and frequently exclude female animals. Here we investigated sex-specific systemic and brain metabolic responses to a standardized KD in mice, highlighting the need to include both sexes. Using the widely used C57BL/6J mouse strain and the Bio-Serv KD, systemic and brain cell metabolism were examined in both sexes. Significant sex-based metabolic differences, probably influenced by hormones, were observed: females were leaner, exhibited higher interindividual variability in weight loss, higher baseline ketone (β-hydroxybutyrate) levels and a faster but less pronounced drop in glycemia compared with males. By contrast, cerebral metabolism appeared stable across sexes, with no significant differences detected in isolated brain cells, suggesting that sex-specific systemic adaptations are counterbalanced at the brain level to maintain functional stability. Regarding brain cell analysis, a lactate gradient from astrocytes to neurons was observed, reflecting preferential astrocytic lactate production and neuronal utilization, along with distinct glucose and glutamine distributions. Overall, our findings validate an animal model of sustained, stable ketosis and emphasize the importance of including both sexes in KD research, providing a foundation for studying sex-specific metabolic adaptations and informing potential personalized dietary strategies.