This study examined the effect of acute exercise and 4 weeks of aerobic training on skeletal muscle gene and protein expression of pro- and anti-angiogenic factors in 14 young male subjects. Training consisted of 60 min of cycling (~ 60% of VO2 max), 3 times/week. Biopsies were obtained from m. v. lateralis before and after training. Muscle interstitial fluid was collected during cycling at week 0 and 4. Training increased (P <0.05) the capillary: fiber ratio and capillary density by 23% and 12%, respectively. The concentration of interstitial vascular endothelial growth factor (VEGF) in response to acute exercise increased similarly (>6-fold; P <0.05) before and after training. Resting protein levels of soluble VEGF receptor-1 in interstitial fluid, and of VEGF, Thrombospondin-1 (TSP-1) and Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in muscle, were unaffected by training, whereas eNOS protein levels in muscle increased by 50% (P <0.05). Before and after training, acute exercise induced a similar increase (P <0.05) in the mRNA level of Angiopoietin 2, matrix metalloproteinase 9 and TSP-1. After training, TIMP-1mRNA content increased with exercise (P <0.05). In conclusion, acute exercise induced a similar increase in the gene-expression of both pro- and anti-angiogenic factors in untrained and trained muscle. We propose that the increase in anti-angiogenic factors with exercise is important for modulation of angiogenesis. The lack of effect of training on basal muscle VEGF protein levels and VEGF secretion during exercise, suggests that increased VEGF levels are not a prerequisite for exercise induced capillary growth in healthy muscle.