Abstract Background Regular physical activity has significant health benefits and can help prevent and manage non-communicable diseases. Intense exercise during training requires diet modulation to support health and performance. Anaerobic sports require high-protein diets, while aerobic sports prefer high-carbohydrate diets. Two-thirds of adult athletes take dietary supplements, and non-professional athletes also use supplements to enhance their appearance. However, dietary stress and imbalanced use of supplements leads the body to different diseases like gastrointestinal diseases and even multifactorial diseases e.g. cancer, cardiovascular diseases, and neurodegenerative diseases. Depending on the specific nutritional intake, supplementations, types of exercises the microbiota can be influenced positively or negatively. A systematic review meta-analysis and a pilot study were conducted to investigate the role of diet and supplementation about the microbial composition of the gut, under different types and intensities of physical activities. Methods The systematic review and meta-analysis were conducted according to the PRISMA guidelines, and the protocol was registered in PROSPERO (Number CRD42024500826). In a pilot study gut microbiota (16S amplicon sequencing), anthropometric data, and information on diet and supplementation, physical activity and sport habits were collected in a sample including professional and non-professional athletes. Results Out of 1318 studies, only 9 met the criteria for inclusion in the meta-analysis. The pilot study’s meta-regression analysis and microbiota analysis highlight the role of physical activity type in changing the Bacillota/Bacteroidota ratio (p = 0.001). Conclusions As gut training becomes more popular among athletes, it is necessary to map interactions between microbiota and different personalized diets, physical activities, and the most popular ergogenic supplements to enhance performance and athletic wellness Key messages • The microbiome can be influenced positively or negatively by the specific dietary intake, types of physical activities and ergogenic supplementations. • To map interactions between microbiota and different diets, physical activities, and popular ergogenic supplements is needed to design new strategies to enhance performance and athletic wellness.