Epidemiological studies indicate females with Facioscapulohumeral Muscular Dystrophy (FSHD) are less affected than males, exhibiting lower clinical severity scores, reduced penetrance, and fewer radiological signs of disease. Likewise, we recently reported evidence for sexual dimorphism in the severity of muscle pathology in the FLExDUX4 murine model of FSHD, with females exhibiting reduced muscle fibrosis, greater running velocities, and greater weight-normalized grip strength than their male littermates. We hypothesize these differences may be caused by higher circulating levels of estrogens, which have beneficial effects on muscle protein synthesis, inflammation, mitochondrial bioenergetics, and sarcolemmal stability. Therefore, we aimed to determine the effect of acutely elevating plasma 17b-estradiol (E2, the major pre-menopausal bioactive estrogen) in un-induced double transgenic (DT) male ACTA1-MCM/FLExDUX4 mice with low levels of leaky skeletal muscle-specific human DUX4 mRNA expression. Laser wounding assays indicated male DT mice (n=4) have significantly reduced sarcolemmal repair capacity compared to their wild type (WT, n=3) littermates (p<0.05), which was significantly improved following oral administration of 1.12 ug E2 daily for 7 days (n=4, p<0.05). Likewise, high resolution mitochondrial respirometry using the Seahorse XFe24 Analyzer indicated DT mice have significantly reduced skeletal muscle oxygen consumption rates, as well as basal, maximal (uncoupled), and ATP-coupled respiration vs WT mice (all p<0.05). E2 administration increased overall oxygen consumption rates (p<0.05) in DT mice, and tended to increase basal and ATP-coupled respiration toward WT levels. Furthermore, DT mice maintain significantly elevated mitochondrial DNA content vs WT mice, which was significantly reduced toward WT levels following E2 administration. These results provide initial evidence that E2 may be beneficial for DUX4-affected skeletal muscle in vivo by improving saroclemmal repair, increasing mitochondrial bioenergetics, and enhancing mitochondrial quality control – molecular defects previously identified in FSHD. Further research is needed to determine if E2 affects DUX4 expression or transcriptional activity in vivo.