Accumulating evidence supports the existence of a tissue microbiota, which may regulate the physiological function of tissues in normal and pathological states. To gain insight into the regulation of tissue-borne bacteria in physiological conditions, we quantified and sequenced the 16S rRNA gene in aseptically collected skeletal muscle and blood samples from eight healthy male individuals subjected to six weeks of endurance training. Potential contamination bias was evaluated and the taxa profiles of each tissue were established. We detected bacterial DNA in skeletal muscle and blood, with background noise levels of detected bacterial DNA considerably lower in control versus tissue samples. In both muscle and blood, Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes were the most prominent phyla. Endurance training changed the content of resident bacterial DNA in skeletal muscle but not in blood, with Pseudomonas being less abundant, and both Staphylococcus and Acinetobacter being more abundant in muscle after exercise. Our results provide evidence that endurance training specifically remodels the bacterial DNA profile of skeletal muscle in healthy young men. Future investigations may shed light on the physiological impact, if any, of training-induced changes in bacterial DNA in skeletal muscle.