The roles of nitric oxide synthase (NOS), reactive oxygen species (ROS) and angiotensin II type 1 receptor (AT₁R) activation in regulating cutaneous vasodilation and sweating during prolonged (≥60 min) exercise are currently unclear. Moreover, it remains to be determined whether fluid replacement (FR) modulates the above thermoeffector responses. To investigate, eleven young men completed 90-min of continuous moderate intensity (46% of VO_2peak) cycling performed at a fixed rate of metabolic heat production of 600 W (No-FR condition). On a separate day, participants completed a second session of the same protocol while receiving FR to offset sweat losses (FR condition). Cutaneous vascular conductance (CVC) and local sweat rate (LSR) were measured at four intradermal microdialysis forearm sites perfused with: (1) lactated Ringer (Control); (2) 10 mM NG-nitro-L-arginine methyl ester (LNAME, NOS inhibition); (3) 10 mM ascorbate (non-selective anti-oxidant); or (4) 4.34 nM Losartan (AT₁R inhibition). Relative to Control (71% CVC_max at both time points), CVC with Ascorbate (80% and 83% CVC_max) was elevated at 60- and 90-min of exercise during FR (both P<0.02) but not at any time during No-FR (all P>0.31). In both conditions, CVC was reduced at end exercise with LNAME (60% CVC_max; both P<0.02), but was not different relative to Control at the Losartan site (76% CVC_max; both P>0.19). LSR did not differ between sites in either condition (all P>0.10). We conclude that NOS regulates cutaneous vasodilation but not sweating, irrespective of FR, and that ROS influence cutaneous vasodilation during prolonged exercise with FR.