This study explores human skeletal patterns rooted in genetic, dietary, and physical activity patterns within a El Japón, an archaeological site on the southern edge of Mexico City. The site was previously inferred to date to the late Postclassic period (1200–1521 CE) based on material remains, especially postclassic ceramics and near absence of colonial ones. Radiocarbon dating and statistical modeling in this research narrow the estimate of El Japón cemetery use to a 90-year period after European contact in the Basin of Mexico, 1550–1640 CE. Dating and modeling facilitate comparison of El Japón’s occupation with the prevalent historical narratives that indicate near complete population relocation of Indigenous communities to larger settlements termed reducciones or congregaciones. Modeling of the radiocarbon results permits discussion of the cemetery sample as a diachronic one to hypothesize and test transformations in lifeways concomitant with Spanish colonization. Interpopulation comparison of dental nonmetric traits permit characterization of the individuals as genetically linked to other local Indigenous populations both predating and postdating European colonization. Radiocarbon and biodistance analysis identify El Japón as a postcontact Indigenous community in a period where an ethnic identity had implications for political stature. These two analyses also contextualize subsequent investigation of subsistence and dietary patterns at El Japón. Cross-sectional properties are employed as proxies of life-long physical activity patterns, and these separate El Japón adults from comparative agricultural and urban European communities of the late Medieval (1250–1500 CE) and early modern periods (1500–1800 CE). Only minor diachronic change took place at El Japón over several decades. Stable isotopic data from bone collagen and bioapatite in El Japón individuals are consistent with precontact populations that relied on local crops and animal protein sources. Continuity in the methods and technologies of agricultural labor and consumption of its products build an image of a small community that persisted in long-rooted lifeways despite the bouts of political and economic change, epidemic health stress, and demographic decline throughout post-Columbian North America.