Incline walking with an external load is a common activity in everyday life. Asymmetrical load carriage can lead to abnormal posture and back pain. Thus, this study aimed to examine the effect of walking uphill with an asymmetrical load in two positions on the spatiotemporal parameters of gait in young adults. Forty-one asymptomatic human volunteers were enrolled in this study. They were asked to walk at a self-selected pace on level and uphill (+5° incline) surfaces carrying a backpack in two asymmetrical positions (hand and shoulder). Spatiotemporal gait parameters were recorded using a photocell device. We observed a significant effect of incline and load position on gait parameters (p < 0.05). Although adaptation to walking on inclines was similar with and without a backpack, adaptation to load position was different when the load was hand-held and shoulder-held. Asymmetric loading with different load locations should be considered an important factor influencing daily gait patterns. In the future, this relationship should be further investigated in terms of pain disorders and postural abnormalities.