http://msystems.asm.org/content/3/3/e00044-18 A Prospective Metagenomic and Metabolomic Analysis of the Impact of Exercise and/or Whey Protein Supplementation on the Gut Microbiome of Sedentary Adults Owen Cronin, Wiley Barton, Peter Skuse, Nicholas C. Penney, Isabel Garcia-Perez, Eileen F. Murphy, Trevor Woods, Helena Nugent, Aine Fanning, Silvia Melgar, Eanna C. Falvey, Elaine Holmes, Paul D. Cotter, Orla O’Sullivan, Michael G. Molloy, Fergus Shanahan ABSTRACT Many components of modern living exert influence on the resident intestinal microbiota of humans with resultant impact on host health. For example, exercise-associated changes in the diversity, composition, and functional profiles of microbial populations in the gut have been described in cross-sectional studies of habitual athletes. However, this relationship is also affected by changes in diet, such as changes in dietary and supplementary protein consumption, that coincide with exercise. To determine whether increasing physical activity and/or increased protein intake modulates gut microbial composition and function, we prospectively challenged healthy but sedentary adults with a short-term exercise regime, with and without concurrent daily whey protein consumption. Metagenomics- and metabolomics-based assessments demonstrated modest changes in gut microbial composition and function following increases in physical activity. Significant changes in the diversity of the gut virome were evident in participants receiving daily whey protein supplementation. Results indicate that improved body composition with exercise is not dependent on major changes in the diversity of microbial populations in the gut. The diverse microbial characteristics previously observed in long-term habitual athletes may be a later response to exercise and fitness improvement. IMPORTANCE The gut microbiota of humans is a critical component of functional development and subsequent health. It is important to understand the lifestyle and dietary factors that affect the gut microbiome and what impact these factors may have. Animal studies suggest that exercise can directly affect the gut microbiota, and elite athletes demonstrate unique beneficial and diverse gut microbiome characteristics. These characteristics are associated with levels of protein consumption and levels of physical activity. The results of this study show that increasing the fitness levels of physically inactive humans leads to modest but detectable changes in gut microbiota characteristics. For the first time, we show that regular whey protein intake leads to significant alterations to the composition of the gut virome.