Abstract Background Gut microbiota is considered to have a great impact on human health and disease. While it is widely recognized that the gut microbiota of healthy individuals differs from those with obesity, inflammatory bowel disease, metabolic syndrome, and other chronic diseases, the alterations of gut microbiota with physical activity are not fully understood. Accordingly, we performed this systematic review to address the question regarding the effects of mild and intense exercise on the gut microbiota in humans. Methods The comparative analyses of gut microbiota were conducted following the PRISMA protocol to determine the differences in the active vs. non-active individuals (phenotypes) (n = 11), including the influence of physical activity intervention on the human gut microbiota (n = 13); the differences in the gut microbiota of athletes vs. non-athletes (n = 8); and the microbiota status at different stages of athletic performance or intervention (n = 7), with various of physical activities, sport disciplines, and activity duration. Literature searches were completed using four databases: PubMed, Web of Science, Scopus, and EBSCO, and 2090 articles were retrieved by using appropriate keywords. The low heterogeneity of the studies hasn’t allowed us to prepare a meta-analysis. After excluding 2052 articles, we ultimately selected 38 articles that met the eligibility criteria for this review. Results The data analyses revealed that in non-athletes rising physical activity markedly influenced the relative abundance of short-chain fatty acid (SCFA). Aerobic training that lasted 60 min, and physical activity that characterized 60% HRmax or more also influenced beta diversity indexes. The results showed that athletes harbor a more diverse type of intestinal microflora than non-athletes, but with a relatively reduced abundance of SCFA- and lactic acid-producing bacteria, thereby suggesting an adverse effect of intense exercise on the population of gut microbiota. Conclusion It is concluded that the level of physical activity modulates the gastrointestinal microbiota in humans. For a long period, increasing the intensity and volume of exercise may lead to gut dysbiosis. Perhaps, proper supplementation should be considered to keep gut microbiota in large biodiversity and richness, especially under unfavorable gut conditions associated with intense exercise. Trial registration Prospero CRD42021264064.