Whether the use of pre-exercise hyperhydration could improve the performance of athletes who do not hydrate sufficiently during prolonged exercise is still unknown. We therefore compared the effects of pre-exercise hyperhydration and pre-exercise euhydration on endurance capacity, peak power output and selected components of the cardiovascular and thermoregulatory systems during prolonged cycling. Using a randomized, crossover experimental design, 6 endurance-trained subjects underwent a pre-exercise hyperhydration (26 ml of water·kg body mass⁻¹ with 1.2 g glycerol·kg body mass⁻¹) or pre-exercise euhydration period of 80 min, followed by 2 h of cycling at 65% maximal oxygen consumption (VO_2max) (26–27°C) that were interspersed by 5, 2-min intervals performed at 80% VO_2max. Following the 2 h cycling exercise, subjects underwent an incremental cycling test to exhaustion. Pre-exercise hyperhydration increased body water by 16.1±2.2 ml·kg body mass⁻¹. During exercise, subjects received 12.5 ml of sports drink·kg body mass⁻¹. With pre-exercise hyperhydration and pre-exercise euhydration, respectively, fluid ingestion during exercise replaced 31.0±2.9% and 37.1±6.8% of sweat losses (p>0.05). Body mass loss at the end of exercise reached 1.7±0.3% with pre-exercise hyperhydration and 3.3±0.4% with pre-exercise euhydration (p<0.05). During the 2 h of cycling, pre-exercise hyperhydration significantly decreased heart rate and perceived thirst, but rectal temperature, sweat rate, perceived exertion and perceived heat-stress did not differ between conditions. Pre-exercise hyperhydration significantly increased time to exhaustion and peak power output, compared with pre-exercise euhydration. We conclude that pre-exercise hyperhydration improves endurance capacity and peak power output and decreases heart rate and thirst sensation, but does not reduce rectal temperature during 2 h of moderate to intense cycling in a moderate environment when fluid consumption is 33% of sweat losses.