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Background Prior operational activities such as marching in diverse environments, with heavy backloads may cause early fatigue and reduce the unit’s readiness. The purpose of this preliminary study was to evaluate the effect of 10-kilometer (km) march on selected, military oriented, physiological and cognitive performances. Eight healthy young males (age 25 ± 3 years) performed a series of cognitive and physiological tests, first without any prior physiological strain and then after a 10 km march in comfort laboratory conditions (24°C, 50%RH) consisting a 5 km/h speed and 2-6% incline with backload weighing 30% of their body weight. Results We found that the subjects’ time to exhaustion (TTE) after the march decreased by 27% with no changes in anaerobic performance. Cognitive performance showed a significant (20%) reduction in accuracy and a tendency to reduce reaction time after the march. Conclusions We conclude that a moderate-intensity march under relatively comfort environmental conditions may differently decrease selected military related physical and cognitive abilities. This phenomenon is probably associated with the type and intensity of the pre-mission physical activity and the magnitude of the associated mental fatigue. We suggest that quantifying these effects, as was presented in this preliminary study, by adopting this practical scientific approach would assist in preserving the soldiers’ performance and health during training and military operations.