Exercise promotes brain plasticity partly by stimulating increases in mature brain-derived neurotrophic factor (mBDNF), but the role of the pro-BDNF isoform in the regulation of BDNF metabolism in humans is unknown. We quantified the expression of pro-BDNF and mBDNF in human skeletal muscle and plasma at rest, after acute exercise (+/- lactate infusion), and after fasting. Pro-BDNF and mBDNF were analyzed with immunoblotting, enzyme-linked immunosorbent assay, immunohistochemistry, and quantitative polymerase chain reaction. Pro-BDNF was consistently and clearly detected in skeletal muscle (40-250 pg mg-1 dry muscle), whereas mBDNF was not. All methods showed a 4-fold greater pro-BDNF expression in type I muscle fibers compared to type II fibers. Exercise resulted in elevated plasma levels of mBDNF (55%) and pro-BDNF (20%), as well as muscle levels of pro-BDNF (∼10%, all P < 0.05). Lactate infusion during exercise induced a significantly greater increase in plasma mBDNF (115%, P < 0.05) compared to control (saline infusion), with no effect on pro-BDNF levels in plasma or muscle. A 3-day fast resulted in a small increase in plasma pro-BDNF (∼10%, P < 0.05), with no effect on mBDNF. Pro-BDNF is highly expressed in human skeletal muscle, particularly in type I fibers, and is increased after exercise. While exercising with higher lactate augmented levels of plasma mBDNF, exercise-mediated increases in circulating mBDNF likely derive partly from release and cleavage of pro-BDNF from skeletal muscle, and partly from neural and other tissues. These findings have implications for preclinical and clinical work related to a wide range of neurological disorders such as Alzheimer's, clinical depression, and amyotrophic lateral sclerosis.