<p>Maximal fat oxidation (MFO) and its corresponding exercise intensity (FATmax) seem to be reduced in individuals with obesity and cardio-metabolic diseases, showing a direct correlation with insulin resistance or sensitivity, muscle oxidative capacity, and adipose tissue lipolysis. In this review, we conducted a systematic search of the PubMed, Scopus, and Web of Science databases, summarizing the findings from 11 studies that evaluated the reproducibility of MFO and FATmax in both healthy individuals and people with obesity. Furthermore, we examine whether the reproducibility of these biomarkers is related to biological or methodological factors, underscoring the importance of standardizing pre-test macronutrient intake and physical activity levels. To further contribute to a standardized assessment of MFO and FATmax, this paper also discusses which exercise protocols and analytical procedures can be applied to minimize its measurement error. Finally, based on the accuracy of different gas analyzers for calculating substrate oxidation at FATmax, this review debates whether the reported day-to-day variation of these biomarkers may only represent the measurement error of these devices. Practical applications of MFO and FATmax reproducibility in the context of scientific research and exercise prescription are also provided, preventing biased hypothesis and theoretical models regarding fat oxidation capacity and cardio-metabolic health.</p>