This in vivo study presents information to assist in the understanding of metatarsal stress fracture etiology. The aims were (a) to provide a fundamental description of loading patterns of the second metatarsal (MTII) during barefoot walking, and (b) to investigate the hypothesis that MTII dorsal strain increases with fatigue and external carrying load. Dorsal MTII strain was measured in vivo under local anaesthetic with an instrumented staple in eight subjects. Experimental conditions were external loading with a 20 kg backpack and pre- and post-fatigue. M. flexor digitorum longus electromyography tentatively indicated fatigue after an extended walking treatment. A reproducible, cyclic temporal pattern of dorsal MTII surface deformation was described. Mean peak compression and tension strains in unloaded barefoot walking were −1534±636 and 363±359 με, respectively. Mean peak compression strain rate (SR) was −4165±1233 με/s. Compression strain increased significantly (α=0.05) both with the addition of the backpack and post-fatigue while maximum tension decreased significantly post-fatigue. SR increased significantly with the addition of the backpack. The highest plantar force time integrals were recorded underneath the heads of metatarsals II–V for all conditions (1561 N s pre-fatigue, without backpack;​ 2123 N s post, with). EMG and plantar pressure data presented a comprehensive description of biomechanical parameters influencing dorsal MTII deformation and alterations in strain following two experimental conditions were suggested as contributing factors in the pathogenesis of metatarsal stress fractures.