The aim of this study was to determine the effect of vascularized fibula free flap (VFFF) harvest on gait variables during the six-minute walk test (6MWT). Eleven patients who had undergone VFFF harvest and 11 healthy peers participated in this case-control study. The main evaluation consisted of the collection of gait variables using the GAITRite system during three periods of the 6MWT: beginning (0-1min), middle (2:30-3:30min), and end (5-6min). The 6MWT was significantly shorter in the VFFF group than in the reference group (-31%, P<0.001). Most gait variables differed significantly between the groups for each period (P-value range 0.04-0.0004), including toe in/out on the operated side (P-value range 0.005-0.01). The increase in toe in/out on the operated side suggests a functional modification caused by an imbalance of the agonist-antagonist muscles. On comparison of the different periods, gait velocity decreased between the beginning and middle periods and increased between the middle and end periods in both groups. However, a significantly lower velocity between the beginning and end periods was found only for the VFFF group (P=0.026), suggesting an alteration in physical management. In conclusion, these results suggest that VFFF harvesting could alter gait and joint integrity.