Objectives To examine the femoral midshaft morphological characteristics in hunter‐gathering Natufian and farming Pre‐pottery Neolithic (PPN) populations in the southern Levant and relate these to changes in mobility, physical stress, and diet. Materials and Methods 32 Natufian, 41 PPNB, and 26 PPNC femora, dating from 14,900 to 8,250 cal BP, were studied. Femoral diaphyseal cross‐sectional images were obtained from CT scans. Dedicated software was used to measure cross‐sectional breadths, areas, cortical bone thickness, rigidity, and strength. Results Two general temporal trends in femoral bone architecture were observed: (1) a continuous decline in the relative amount of bone tissue (cortical area/total area) due to expansion of the medullary cavity and (2) an increase in circularity (decrease in anteroposterior/mediolateral ratios) together with an overall decline in bone rigidity and strength, mainly apparent in the later PPNC. The first trend suggests a gradual decline in nutritional quality and health continuing from the Natufian through the late Neolithic. The second trend is interpreted as a result of increased sedentism with the full establishment of agriculture. Discussion The transition to food production in the southern Levant was accompanied by reduced physical stress and mobility, with the most marked effects occurring toward the end of the PPN with increasing sedentism. Deterioration of nutrition and health also occurred, but more continuously from the beginning of the PPN. Thus, environmental changes associated with the agricultural transition in this region of the world were gradual and prolonged, with direct dietary effects more apparent earlier than reductions in mobility. Am J Phys Anthropol 161:26–36, 2016. © 2016 Wiley Periodicals, Inc.