Principal strains and their orientation, determined from in vivo and in situ strains recorded from the lateral cortical surface of the calcaneus of potoroos (a small marsupial) during treadmill exercise and tension applied via the Achilles tendon, were compared with the underlying trabecular architecture and its alignment to test Wolff s “trajectorial theory” of trabecular alignment. In vivo and in situ principal compressive strains (−800 to −2000 μe) were found to be aligned (mean 161 ± 7°) close to the preferred alignment (160°) of underlying trabeculae within the calcaneal metaphysis [a second trabecular arcade was closely aligned (70°) with the direction (71°) of principal tensile strain]. This finding represents quantitative verification of Wolffs trajectorial theory of trabecular alignment. These trabecular alignments, as measured by trabecular anisotropy (TbAn, the ratio of horizontal:​ vertical intercepts), remained unchanged (p > 0.05) after 8 weeks of disuse. However, trabecular bone volume fraction (BV/TV, −35%), trabecular thickness (TbTh, −25%), and trabecular number (TbN, −16%) were reduced for the tenotomized calcaneii relative to their contralateral controls (p < 0.001 to < 0.003). The reduction in trabecular number was associated with a corresponding increase in trabecular spacing (TbSp, +30%). Together, these results suggest that once trabecular alignment is established during growth (along the directions of principal strain during locomotion), it is not altered when functional strains are removed.