This study was to describe and compare the physiological demands of ultra-endurance cyclists during a 24 h cycling relay race. Eleven male athletes (means +/- SD: 34.8 +/- 5.6 years; 71.6 +/- 4.9 kg; 174.6 +/- 7.3 cm; BMI 23.5 +/- 0.5 kg/m2; VO2 max: 66.0 +/- 6.4 ml/kg/min) participated in the study; eight in teams with a format of four riders (4C) and three in teams with six riders (6C). To investigate exercise intensity, heart rate (HR) was recorded while cycling using portable telemetric monitors. Three different exercise intensities were defined according to the reference HR values obtained during a pre race laboratory incremental VO2 max test: Zone I (< anaerobic threshold [AT]), Zone II (between AT and the respiratory compensation point [RCP]), Zone III (> RCP). Total volume and intensity were integrated as a single variable (training impulse: TRIMP). The score for TRIMP in each zone was computed by multiplying the accumulated duration in this zone by a multiplier for this particular zone of exercise intensity. The average intensity did not differ between cyclists in 4C (means +/- SD; 4C: 87 +/- 3 HRmax) and 6C (87 +/- 1% of HRmax), despite the higher volume performed by 4C (means +/- SD; 4C: 361 +/- 65; 6C: 242 +/- 25 per min; P = 0.012). These differences in total exercise volume significantly affected the values TRIMP accumulated (means +/- SD; 4C: 801 +/- 98, confidence interval [CI] 95%: 719 - 884; 6C: 513 +/- 25, CI 95%: 451 - 575; P = 0.012). The ultra-endurance threshold of 4C and 6C athletes lies at about 87% of HRmax for both. Although the intensity profile was similar, the TRIMP values differed significantly as a consequence of the higher volume performed by the 4C cyclists.