We compared the effect of glycerol-induced hyperhydration (GIH) to that of water-induced hyperhydration (WIH) on cardiovascular and thermoregulatory functions and endurance performance (EP) during prolonged cycling in a temperate climate in subjects consuming fluid during exercise. At weekly intervals, 6 trained male subjects ingested, in a randomized, double-blind, counterbalanced fashion, either a glycerol (1.2 g glycerol/kg bodyweight (BW) with 26 mL/kg BW of water-aspartame-flavored fluid) or placebo solution (water-aspartame-flavored fluid only) over a 2 h period. Subjects then performed 2 h of cycling at 66% of the maximal oxygen consumption (VO(2) max) and 25 degrees C while drinking 500 mL/h of sports drink, which was followed by a step-incremented cycling test to exhaustion. Levels of hyperhydration did not differ significantly between treatments before exercise. During exercise, GIH significantly reduced urine production by 246 mL. GIH did not increase sweat rate nor did it decrease heart rate, rectal temperature, or perceived exertion during exercise as compared with WIH. EP was not significantly different between treatments. Neither treatment induced undesirable side effects. It is concluded that, compared with WIH, GIH decreases urine production, but does not improve cardiovascular orthermoregulatory functions, nor does it improve EP during 2 h of cycling in a 25 degrees C environment in trained athletes consuming 500 mL/h of fluid during exercise.