The effect of a prolonged running trial on the energy cost of running (C_r) during a 60-km ultramarathon simulation at the pace of a 100-km competition was investigated in 13 men (40.8 ± 5.6 y, 70.7 ± 5.5 kg, 177.5 ± 4.5 cm) and 5 women (40.4 ± 2.3 y, 53.7 ± 4.4 kg, 162.4 ± 4.8 cm) who participated in a 60-km trial consisting of 3 consecutive 20-km laps. Oxygen uptake (VO₂) at steady state was determined at constant speed before the test and at the end of each lap; stride length (SL) and frequency and contact time were measured at the same time points; serum creatine kinase (S-CPK) was measured before and at the end of the test. C_r in J · kg⁻¹ · m⁻¹, as calculated from VO_2ss and respiratory-exchange ratio, did not increase with distance. SL significantly decreased with distance. The net increase in S-CPK was linearly related with the percentage increase of C_r observed during the trial. It is concluded that, in spite of increased S-CPK, this effort was not able to elicit any peripheral or central fatigue or biomechanical adaptation leading to any modification of C_r.