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The southward expansion of East Asian farmers profoundly influenced the social evolution of Southeast Asia by introducing cereal agriculture. However, the timing and routes of cereal expansion in key regions are unclear due to limited empirical evidence. Here we report macrofossil, microfossil, multiple isotopic (C/N/Sr/O) and paleoproteomic data directly from radiocarbon-dated human samples, which were unearthed from a site in Xingyi in central Yunnan and which date between 7000 and 3300 a BP. Dietary isotopes reveal the earliest arrival of millet ca. 4900 a BP, and greater reliance on plant and animal agriculture was indicated between 3800 and 3300 a BP. The dietary differences between hunter-gatherer and agricultural groups are also evident in the metabolic and immune system proteins analysed from their skeletal remains. The results of paleoproteomic analysis indicate that humans had divergent biological adaptations, with and without farming. The combined application of isotopes, archaeobotanical data and proteomics provides a new approach to documenting dietary and health changes across major subsistence transitions., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2023 Science China Press. Published by Elsevier B.V. All rights reserved.)