This study highlights the need for standardized ketogenic diet (KD) research that includes both sexes, as most previous studies have focused on male animals. Using the most widely used strain in biomedical research, the genetically uniform C57BL/6J mice, two KD suppliers (Safe and Bio-Serv) were compared to assess the impact of diet composition on metabolic outcomes. Safe’s KD induced ketosis initially but caused metabolic disruptions due to stratification in the diet's composition. Bio-Serv’s KD consistently maintained ketosis and was preferred for standardization. Significant sex-based differences were found, likely due to the influence of hormones. Brain cell analysis revealed a lactate gradient from astrocytes to neurons and differences in glucose and glutamine distribution, though limited by sample size. The study reinforces the importance of including both sexes in metabolic research and maintaining diet consistency to better understand hormonal effects and long-term health impacts of the KD.