Ageing is associated with impaired endothelium-derived nitric oxide (NO) function in human microvessels. We investigated the impact of cardiorespiratory fitness and exercise training on physiological and pharmacological NO-mediated microvascular responses in older subjects. NO-mediated vasodilation was examined in young, older sedentary and older fit subjects who had 2 microdialysis fibres embedded into the skin on the ventral aspect of the forearm and laser Doppler probes placed over these sites. Both sites were then heated to 42°C, with Ringers solution infused in one probe and N-nitro-L-arginine methyl ester (LNAME) through the second. In another study, 3 doses of ACh were infused in the presence or absence of LNAME in similar subjects. The older sedentary subjects then undertook exercise training, with repeat studies at 12 and 24 weeks. The NO component of the heat-induced rise in cutaneous vascular conductance (CVC) was diminished in the older sedentary subjects after 30 minutes of prolonged heating at 42ºC (26.9±3.9%), compared to older fit (46.2±7.0, P<0.05) and young subjects (41.2±5.2%, P<0.05), whereas exercise training in the older sedentary group enhanced NO-vasodilator function in response to incremental heating (P<0.05). Similarly, the NO contribution to ACh responses was impaired in the older sedentary versus older fit subjects (low dose 3.2±1.3 vs 6.6±1.3; mid dose 11.4±2.4 vs 21.6±4.5; high dose=35.2±6.0 vs 52.6±7.9%CVCmax, P<0.05) and training reversed this (12 weeks: 13.7±3.6, 28.9±5.3, 56.1±3.9. P<0.05). These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents age-related decline in indices of physiological and pharmacological microvascular NO-mediated vasodilator function. Since higher levels of NO confer anti-atherogenic benefit, this study has potential implications for the prevention of microvascular dysfunction in humans.