The main purpose of the present study was to compare the reference metabolic equivalent (MET) value and observed resting oxygen uptake (VO2) for defining cardiorespiratory fitness (VO2max) and characterizing the energy cost of treadmill running. A heterogeneous cohort of 114 healthy men volunteered to participate. In Part 1 of the study, 114 men [mean ± SD, age: 24 ± 5 years; height: 177.1 ± 7.9 cm; body mass: 75.0 ± 10.0 kg] visited the laboratory twice for assessment of resting and maximal VO2 values to compare the reference MET value vs. observed resting VO2 and to investigate the association between resting VO2 and VO2max. In Part 2, 14 of the 114 men visited the laboratory once more to perform a 30-min bout of running at 8.0 km∙h-1/8.3 METs. The mean observed resting VO2 of 3.26 mL•kg-1•min-1 was lower than the reference MET value of 3.5 mL•kg-1•min-1 (P < 0.001). Resting and maximal VO2 values relative to total body mass and fat-free mass were positively correlated (R = 0.71 and 0.60, respectively; P < 0.001). The maximal MET and energy cost of treadmill running were consequently underestimated when calculated using the reference MET value only for those with low VO2max (P = 0.005 to P < 0.001). In conclusion, the reference MET value considerably overestimated observed resting VO2 in men with low VO2max, resulting in underestimations of the maximal MET, exercise intensity prescription, and the energy cost of running.