A comprehensive measurement analysis of soldiers’ gait and motion during prolonged loaded March is vital in order to analyse the consistency of soldiers’ performance during combat and training. Prolonged loaded March has been reported to cause fatigue and overuse injuries to the trunk and lower extremity, thus inhibiting soldiers to attain their optimal performance. A motion capture system, considered as the reference standard in motion analysis, is used in a controlled environment for this research. Data were collected from 10 healthy male soldiers (26.3 ± 5.8 years old), performing load March on treadmill at 6.4 km/h (1% elevation) while carrying 15 kg loaded military backpack for 30 min. The study was able to address the complete 3-dimensional measurement analysis of soldiers’ gait (kinematics and spatio-temporal data), heart rate and their perceived exertion. Different kinematic features such as angular changes and range of motion in critical joints; ankle, knee, hip, pelvis and trunk, integrated with the spatio-temporal features; physiological data such as heart rate (HR) and subjective responses such as rating of perceived exertion (RPE) at critically important gait events were examined. The results prove significant changes (p < 0.05) in kinematic features such as angular changes of ankle joint at frontal plane during toe-off and angular changes of knee joint at transverse plane during all gait events, including significant increase in HR and RPE values towards the end of the load carriage trial. The study was able to identify which critical joint at a particular plane and gait event that was affected first and most significantly by the load carriage trial. It has also shown that due to the load weight, speed and duration of the task that is routinely used in military training and soldier fitness regime, subjects were able to consistently replicate their kinematic patterns throughout the experimental task.