This study investigates the comprehensive physiological, biomechanical, and thermographic responses of male athletes during an ultra-endurance race, the Santander Four Days (S4D). Involving a 160 km race over four consecutive days with a 10 kg backpack, the study focuses on key aspects such as body mass, cortical arousal, handgrip strength, heart-rate variability, hydration status, blood glucose and lactate concentrations, and thermographic responses. The results indicate changes in heart-rate variability, indicating increased cardiovascular strain, consistent neuromuscular performance, significant body-weight reduction possibly due to dehydration and energy use, stable pH and glucose, but increased protein in urine suggesting renal stress, and varied body temperatures reflecting physical exertion and environmental factors. These findings highlight the body’s adaptive mechanisms and the importance of specialized training and recovery strategies in such physically demanding events.