2019 Background: Physical exercise, such as walking, is imperative to older adults. However, long-distance walking may increase walking instability which exposes them to some fall risks. Objective: To evaluate the influence of long-distance walking on gait asymmetry and variability of older adults. Method: Sixteen physically active older adults were instructed to walk on a treadmill for a total of 60 min. Gait experiments were conducted over-ground at the baseline (before treadmill-walk), after first 30 min (30-min) and second 30 min (60-min) of the walk. In addition to spatiotemporal parameters, median absolute deviation of the joint angular velocity was measured to evaluate gait asymmetry and gait variability. Findings: There were significant differences in the overall asymmetry index among the three time instances (Partial η2 = 0.77, p < .05), predominantly contributed by the ankle (Partial η2 = 0.31, p < .017). Long-distance walking significantly increased the average and maximum median absolute deviation of the ankle at both sides (W ≥ 0.19, p < .05), and knee at the non-dominant side (W = 0.44, p < .05). Interpretation. At 30-min, the older adults demonstrated a significantly higher asymmetry and variability at the ankle, which implied higher instability. Continue walking for an additional 30 min (60-min) further increased variability of the non-dominant limb at the knee joint. Walking for 30 min or more could significantly reduce walking stability.