The aim of this study was to investigate whether variation in gait-related parameters among healthy participants could help detect gait abnormalities. In total, 36 participants (21 men, 15 women; mean age, 35.7 ± 9.9 years) performed a 10-m walk six times while wearing a tri-axial accelerometer fixed at the L3 level. A second walk was performed ≥1 month after the first (mean interval, 49.6 ± 7.6 days). From each 10-m data set, the following nine gait-related parameters were automatically calculated: assessment time, number of steps, stride time, cadence, ground force reaction, step time, coefficient of variation (CV) of step time, velocity, and step length. Six repeated measurement values were averaged for each gait parameter. In addition, for each gait parameter, the difference between the first and second assessments was statistically examined, and the intraclass correlation coefficient (ICC) was calculated with the level of significance set at p < 0.05. Only the CV of step time showed a significant difference between the first and second assessments (p = 0.0188). The CV of step time also showed the lowest ICC, at <0.50 (0.425), among all parameters. Test–retest results of gait assessment using a tri-axial accelerometer showed sufficient reproducibility in terms of the clinical evaluation of all parameters except the CV of step time.