In a test of hypothalamic-pituitary-adrenal (HPA) cortical and hypothalamic-pituitary-gonadal (HPG) interaction during familiar and novel stress, we previously reported that treadmill exercise training led to blunted plasma adrenocorticotrophin (ACTH) response to acute treadmill running but a hyper-responsiveness of ACTH after novel immobilization. In this follow-up analysis, we examined whether those results might be plausibly explained by a similar effect of treadmill exercise training on increased levels of norepinephrine (NE) in hypothalamic and limbic brain regions which synergize to modulate the release of ACTH during stress. Ovariectomized Sprague-Dawley rats that had been exercise trained by treadmill running or remained sedentary for 6 weeks received intramuscular injections of estradiol benzoate (Eb) or sesame oil on each of 3 days prior to 15 min of familiar treadmill running or novel immobilization. Treadmill exercise training, regardless of Eb treatment or type of stress, increased NE levels in the paraventricular (PVN), arcuate, medial preoptic, and ventromedial areas of the hypothalamus and protected against depletion of NE in the locus coeruleus, amygdala, and hippocampus. We conclude that treadmill exercise training has a hyperadrenergic effect in brain areas that modulate hypothalamic regulation of ACTH release during stress that is independent of HPA-HPG interaction and novelty of the stressor. To help elucidate these findings, the effects of treadmill exercise training on A1-A2 nuclei which innervate the PVN and their relationship with the limbic and hypothalamic responses we report require study.