Gait variability is an important measure in clinical settings to diagnose older individuals with fall risk. This study examines whether a familiarization trial improves test–retest reliability of gait variability. Twenty-two older participants walked twice at 1 day and twice 7 days later. The standard deviations of stride length, swing time, stance time, stride time and minimum foot clearance were calculated. The test–retest reliability of (1) between-day comparison of the first trials and (2) between-day comparison of the second trials of all gait variability measures was quantified with the intraclass-correlation coefficient (ICC), the smallest detectable differences (SDD) and the bias and the limits of agreement (LoA). The between-day comparison of the second trials per day showed higher ICC values, lower LoA values and lower SDD values in all analyzed parameters. Our data suggest that the reliability of gait variability in an older population can be considerably improved just with the aid of one familiarization trial.