The effects of footwear and inclination on running biomechanics over short intervals are well documented. Although recognized that exercise duration can impact running biomechanics, it remains unclear how biomechanics change over time when running in minimalist shoes and on slopes. Our aims were to describe these biomechanical changes during a 50-minute run and compare them to those observed in standard shoes. Thirteen trained recreational male runners ran 50 minutes at 65% of their maximal aerobic velocity on a treadmill, once in minimalist shoes and once in standard shoes, 1 week apart in a random order. The 50-minute trial was divided into 5-minute segments of running at 0%, +5%, and -5% of treadmill incline sequentially. Data were collected using photocells, high-speed video cameras, and plantar-pressure insoles. At 0% incline, runners exhibited reduced leg stiffness and plantar flexion angles at foot strike and lower plantar pressure at the forefoot and toes in minimalist shoes from minute 34 of the protocol onward. However, only reduced plantar pressure at the toes was observed in standard shoes. Overall, similar biomechanical changes with increased exercise time were observed on the uphill and downhill inclines. The results might be due to the unfamiliarity of subjects to running in minimalist shoes.