The objective of this study was to determine whether one session of targeted locomotor training can induce measurable improvements in the post-stroke gait impairments. Thirteen individuals with chronic post-stroke hemiparesis participated in one locomotor training session combining fast treadmill training and functional electrical stimulation (FES) of ankle dorsi- and plantar-flexor muscles. Three dimensional gait analysis was performed to assess within-session changes (after versus before training) in gait biomechanics at the subject's self-selected speed without FES. Our results showed that one session of locomotor training resulted in significant improvements in peak anterior ground reaction force (AGRF) and AGRF integral for the paretic leg. Additionally, individual subject data showed that a majority of study participants demonstrated improvements in the primary outcome variables following the training session. This study demonstrates, for the first time, that a single session of intense, targeted post-stroke locomotor retraining can induce significant improvements in post-stroke gait biomechanics. We posit that the within-session changes induced by a single exposure to gait training can be used to predict whether an individual is responsive to a particular gait intervention, and aid with the development of individualized gait retraining strategies. Future studies are needed to determine whether these single-session improvements in biomechanics are accompanied by short-term changes in corticospinal excitability, and whether single-session responses can serve as predictors for the longer-term effects of the intervention with other targeted gait interventions.