Objective To investigate the effects of unevenly-distributed backpack loads on human physiological parameters as compared to the traditional centrally-placed load at the backpack bottom. Methods For the unevenly-distributed load mode, the backpack was divided into four spaces including top right, top left, bottom right and bottom left. The loads were divided into two parts proportionally (1/2∶1/2, 1/4∶3/4 and 3/4∶1/4) and placed at the bottom left and top right of the backpack, respectively. The remaining two spaces were filled with the plastic foam. Ten healthy volunteers performed 30-min walking trials on the treadmill at the speed of 1.1 m/s with the backpack load equal to 10% body weight (BW). The changes of subjects’ body posture, muscle fatigue, heart rate, blood pressure before and after the trial, as well as the electromyography (EMG) changes at 0, 5, 10, 15 min after the trial were measured by the Bortec AMT-8 and the NDI Optotrak Certus. Results The muscle fatigue of right upper trapezius and forward-leaning angle increased as the loads at top right of the backpack increased. However, the muscle fatigue in the new mode of backpack load distribution showed no significant differences as compared to the traditional mode under the same backpack loads. The new mode with the load ratio of 3/4∶1/4 had no significant effects on posture. The new mode with the load ratio of 1/2∶1/2 could contribute to the decrease of heart rate and blood pressure as compared to the traditional mode. Conclusions Adopting a new and more reasonable load distribution mode can guarantee the body posture unaffected by the backpack loads, and decrease heart rate and blood pressure. Therefore, the cross and evenly distributed load mode is recommended for the backpack design.