IMPORTANCE The chimeric anterolateral thigh osteomyocutaneous (ALTO) free flap is a recently described microvascular option for head and neck osseous defects associated with complex soft-tissue requirements. To date, the association of ALTO flap harvest with femur structural integrity and the need for routine prophylactic fixation following harvest has been incompletely described. OBJECTIVE To investigate the association of ALTO flap harvest, with and without prophylactic fixation, on femur structural integrity as measured by 4-point bend and torsional biomechanical testing. DESIGN AND SETTING At a research laboratory, 24 synthetic fourth-generation composite femurs with validated biomechanical properties underwent 10-cm-long, 30% circumferential osteotomies at the proximal middle third of the femur; 6 femurs served as controls. Osteotomized femurs with and without fixation underwent torsional and 4-point bend biomechanical testing. Femur fixation consisted of intramedullary nail and distal interlock screw placement. MAIN OUTCOMES AND MEASURES Force and torque to fracture (expressed in kilonewtons [kN] and Newton meters [N·m], respectively) were compared between controls, osteotomized femurs without fixation, and osteotomized femurs with fixation. Additional outcome measures included femur stiffness and fracture patterns. RESULTS On posterior to anterior (PA) 4-point bend testing, force to fracture of osteotomized femurs was 22% of controls (mean difference, 8.3 kN; 95% CI, 6.6–10.0 kN). On torsional testing the torque to fracture of osteotomized femurs was 12% of controls (mean difference, 351.1 N·m; 95% CI, 307.1–395.1 N·m). Following fixation there was a 67% improvement in PA force to fracture and a 37% improvement in torque to fracture. However, osteotomized femurs with fixation continued to have a reduced PA force to fracture at 37% of controls (mean difference, 6.8 kN; 95% CI, 4.5–9.2 kN) and torque to fracture at 16% of controls (mean difference, 333.7 N·m; 95% CI, 306.8–360.6 N·m). On torsional testing, all osteotomized femurs developed similar spiral fractures through a corner of the distal osteotomy site. This fracture pattern changed after prophylactic fixation with femurs developing nondisplaced fractures through the proximal osteotomy site. There were no underlying hardware failures during testing of osteotomized femurs with fixation. CONCLUSIONS AND RELEVANCE Anterolateral thigh osteomyocutaneous flap harvest results in significant changes in the structural integrity of the femur. Postoperative stabilization should be strongly considered, with future research directed at investigating the clinical significance of residual biomechanical changes following femur fixation.