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The objective of this study was to investigate the nutritional functions of highland barley subjected to heat-moisture treatment (HMT) and dry heat treatment (DHT) in vitro and in vivo. In vitro test indicated HMT and DHT play part in the reduced glycemic potency of highland barley. Meanwhile, in vivo results showed that thermally-processed highland barley (THB) supplementation significantly decreased the body weight and serum glucose, improved oxidation resistance and altered the composition of gut microbiota. Bifidobacteria, Fusicatenibacter and Desulfovibrio were identified as types of bacteria that might related to the relatively higher content of dietary fiber in THB. The Spearman’s correlation analysis revealed that Fusicatenibacter and Desulfovibrio were positively correlated with T-AOC levels. In addition, the putative metagenomes implicated that THB might regulate the metabolic pathways of gut microbiota …