Stem cells functions are regulated by different factors and non-conding RNAs, such as microRNA. MiRNAsplay an important role in modulating the expression of genes involved in the commitment and differentiation of progenitor cells. MiRNAs are post transcriptional regulators which may be modulated by physical exercise. MiRNAs, by regulating different signaling pathways, play an important role in myogenesis as well as in muscle activity. MiRNAs quantification may be considered for evaluating physical performance or muscle recovery. With the aim to identify specific miRNAs potentially involved in myogenesis and modulated by physical activity, we investigated miRNAs expression following physical performance in Peripheral Blood Mononuclear Cells (PBMCs) and in sera of half marathon (HM) runnners. The effect of runners sera on Myogenesis in in vitro cellular models was also explored. Therefore, we performed Microarray Analysis and Real Time PCR assays, as well as in vitro cell cultures analysis to investigate myogenic differentiation. Our data demonstrated gender-specific expression patterns of PBMC miRNAs before physical performance. In particular, miR223-3p, miR26b-5p, miR150-5p and miR15-5p expression was higher, while miR7a-5p and miR7i-5p expression was lower in females compared to males. After HM, miR152-3p, miR143-3p, miR27a-3p levels increased while miR30b-3p decreased in both females and males: circulating miRNAs mirrored these modulations. Furthermore, we also observed that the addition of post-HM participants sera to cell cultures exerted a positive effect in stimulating myogenesis. In conclusion, our data suggest that physical activity induces the modulation of myogenesis-associated miRNAs in bothfemales and males, despite the gender-associated different expression of certain miRNAs, Noteworthy, these findings might be useful for evaluating potential targets for microRNA based-therapies in diseases affecting the myogenic stem cells population.