Recent studies have demonstrated that paternal stress in rodents can result in modification of the sperm small non-coding (snc) RNA content as well as changes in the offspring behavioural and physiological traits. We recently showed that paternal exposure to corticosterone affected several microRNAs in the sperm of adult mice and had an impact on the offspring behavioural phenotype (Short et al., 2016). Moreover, we now show that environmental stimulation, via environmental enrichment, of male mice also influenced the behavioural and physiological phenotypes of their progeny (Yeshurun et al., 2016). We then aimed to investigate the sncRNA content of the sperm in response to increased physical activity. We collected sperm cells from epididymis of either control mice or mice with induced physical activity. Sperm samples were sequenced and analysed by both genomic reference annotation and by using tDRmapper, which quantifies transfer-derived RNAs. Analysis of the sncRNA profile in sperm from physically active males revealed marked effects on the expression of sncRNAs. Interestingly, all altered transfer-derived RNAs were increased. Further analysis of transfer-derived RNAs by tDRmapper revealed significant changes in tRNA halves (5’-tRHs) and tRNA fragments (3’-tRFs). Subsequently, we performed target prediction of the top 5 significantly altered miRNAs and 5’-tRHs and we tested their gene ontology in the Mouse Genome Informatics (MGI) phenotype using Enrichr. Remarkably, most ontologies were related to brain and behaviour functions for both gene lists. These results show that physical activity significantly altered the sncRNA content of the sperm with a major effect on transfer-derived RNAs. Therefore, our study provides additional evidence for the role of tRNA halves in the epigenetic regulation of the sperm.