Despite the rapid growth of mass-participation road cycling, little is known about the dietary, metabolic and behavioral responses of ultra-endurance cyclists. This investigation describes physiological responses, perceptual ratings, energy balance, and macronutrient intake of 42 men (mean ± SD; age, 38±6 y; height, 179.7±7.1 cm; body mass, 85.85±14.79 kg) and 6 women (age, 41±4 y; height, 168.0±2.9 cm; body mass, 67.32±7.21 kg) during a summer 164-km road cycling event. Measurements were recorded one day before, and on Event Day (10.5 h) at the start (0 km), at two aid stations (52 km and 97 km), and at the finish line (164 km). Ambient temperature was >39.0°C during the final 2 h of exercise. Mean finish times for men (9.1±1.2 h) and women (9.0±0.2 h) were similar, as were mean gastrointestinal temperature [TGI], four hydration biomarkers, and five perceptual (e.g., thermal, thirst, pain) ratings. Male cyclists consumed enough fluids on Event Day (5.91±2.38 L; 49% water) to maintain body mass within 0.76 kg, start to finish, despite a sweat loss of 1.13±0.54 L·h-1 and calculated energy expenditure of 3,115 Kcal·10.5h-1 . However, men voluntarily underconsumed food energy (deficit of 2,594 Kcal, 10.9 MJ), and specific macronutrients (carbohydrates, 106±48 g; protein, 8 ± 7 g; and sodium, 852 ± 531 mg) between 0530 and 1400 h. Also, a few men exhibited extreme final values (i.e., urine specific gravity of 1.035 to 1.038, n=5; body mass loss >4 kg, n=2; TGI, 39.4 and 40.2). We concluded that these findings provide information regarding energy consumption, macronutrient intake, hydration status and the physiological stresses that are unique to ultra-endurance exercise in a hot environment.