Childhood obesity has reached epidemic levels, representing one of the most serious public health concerns associated with metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). There is limited clinical experience for pediatric NAFLD patients and the therapeutic options are very scarce. The aim of this study is to evaluate the benefits of exercise on gut microbiota composition and functionality balance and its repercussion on early obesity and NAFLD onset in an in vivo model. 21 days old male Wistar rats fed with control or high fat diet (HFD) were subjected to a combined aerobic and resistance training protocol. Fecal microbiota was sequenced by Illumina MiSeq system and parameters related to metabolic syndrome, fecal metabolome, intestinal barrier integrity, bile acids metabolism and transport, and gut-liver axis alteration were measured. Exercise decreased HFD-induced body weight gain, metabolic syndrome and hepatic steatosis, as a result of its lipid metabolism modulatory capacity. The gut microbiota composition and functionality were substantially modified as a consequence of diet, age, and exercise intervention. Besides, the training protocol increased Parabacteroides, Bacteroides and Flavobacterium genera which correlated with a beneficial metabolomic profile, while Blautia, Dysgonomonas and Porphyromonas showed an opposite pattern. Exercise effectively counteracts HFD-induced microbial imbalance, leading to intestinal barrier preservation, which in turn prevented gut-liver axis deregulation and improved bile acids homeostasis, determining clinical outcomes of NAFLD. In conclusion, we provide scientific evidences highlighthing the benefits of gut microbiota composition and functionality modulation by physical exercise protocols in the management of early obesity and NAFLD development.