•  Angiogenic regulators respond to acute exercise with different temporal expression patterns (e.g. 2–4 h versus 12–24 h) creating a complex multifaceted response that must be considered in studies using a single time point for post‐exercise analyses. •  In response to chronic training there appears to be a complex coordination in the proteomic responses of both positive and negative angiogenic factors that correspond with training‐induced muscle capillary adaptation, such that altered basal expression and acute responses to exercise appear to withdraw or reduce the stimulus of angiogenic regulators in an expanding capillary bed with active angiogenesis. •  These are the first data to show that nucleolin (a protein responsible for transcriptional processing and transportation of proteins from the cytoplasm to the nucleus) is responsive to acute exercise. We speculate that nucleolin may work in concert with vascular endothelial growth factor‐A (VEGF) and endostatin. •  Temporal responses observed in mice, particularly for VEGF, MMP‐2 and MMP‐9, may not be directly comparable to humans. Abstract  Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro‐angiogenic and anti‐angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor‐A (VEGF), endostatin and nucleolin were increased at 2–4 h (P < 0.05), whereas matrix metalloproteinase (MMP)‐2 was elevated within a 12–24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP‐2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP‐1) was ∼900% greater in 14d‐ and 28d‐trained mice compared to either 5d‐ and 7d‐trained mice (P < 0.05), and tended to increase by ∼180–258% compared to basal control levels (P < 0.10). The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly‐coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.