Background A high incidence of lower leg overuse injury has been observed among military populations. Footwear usage could be promising in reducing the risk of injury from overuse of the lower extremities. The purposeof this study was to identify possible gait-related risk factors for lower leg overuse injurieswhen walking barefoot and in military boots. Methods Cases (n=32) were active-duty infantry soldiers from Latvian Land Forces with a history of overuse injury during the last 6 months of service before entering the study. During the study period, the cases recovered completely from the injury, did not report any functional limits, and were able to participate in all kinds of physical activities. Controls (n=32) of the same age and gender were free of injury in the same time period. Study subjects were instructed to walk on a 5-meter walkway in the gait laboratory. Rearfoot eversion, ankle plantar/dorsiflexion and stride time, length and variability were evaluated for barefoot and shod conditions. Results Gait-related parameters during shod and barefoot conditions differ in both groups. The barefoot stride time and the stride time variability were statistically different between cases and controls. No significant differences were found for stride length and stride time variability among groups. Conditional regression analysis showed that stride time variability during barefoot gait statistically significantly predicted the risk oflower leg overuse injury. ROC analysis showed an AUC of 0.77 (p<0.001; 95% CI 0.648-0.883), a sensitivity of 56%, and aspecificity of 88%, with an optimal cut-off value for stride time variability of 1.95%. Conclusions The risk of injury was independent of thegait-related characteristics during shod gait. Stride time variability has low sensitivity, but if the variability value during the barefoot walk is less than 1.95%, it can be considered a negative risk factor for lower leg overuse injury among infantry soldiers. Trial registration Not applicable.