Purpose. The physiological mechanisms for alterations in oxygen utilization (V˙O2) and the energy cost of running (Cr) during prolonged running are not completely understood, and could be linked with alterations in muscle and cerebral tissue oxygenation. Methods. Eight trained ultramarathon runners (three women; mean ± SD; age 37 ± 7 yr; maximum V˙O2 60 ± 15 mL min-1 kg-1) completed a 6 hr treadmill run (6TR), which consisted of four modules, including periods of moderate (3 min at 10 km h-1, 10-CR) and heavy exercise intensities (6 min at 70% of maximum VO2, HILL), separated by three, 100 min periods of self-paced running (SP). We measured V˙O2, minute ventilation (V˙E), ventilatory efficiency (V˙E:VO2), respiratory exchange ratio (RER), Cr, muscle and cerebral tissue saturation index (TSI) during the modules, and heart rate (HR) and perceived exertion (RPE) during the modules and SP. Results. Participants ran 58.3 ± 10.5 km during 6TR. Speed decreased and HR and RPE increased during SP. Across the modules, HR and V˙O2 increased (10-CR), and RER decreased (10-CR and HILL). There were no significant changes in V˙E, V˙E:V˙O2, Cr, TSI and RPE across the modules. Conclusions. In the context of positive pacing (decreasing speed), increased cardiac drift and perceived exertion over the 6TR, we observed increased RER and increased HR at moderate and heavy exercise intensity, increased V˙O2 at moderate intensity, and no effect of exercise duration on ventilatory efficiency, energy cost of running and tissue oxygenation.