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To investigate the reliability and validity of a functional performance test in patients with chronic ankle instability (CAI).Cross-sectional study.All tests were conducted in the practice room of the physical therapy department of the Vrije Universiteit Brussel.Twenty-nine healthy subjects and 29 patients with CAI were selected. For the main outcome measurements, participants performed a multiple hop test two times, within a 1-week time interval. Subjects hopped on 10 different tape markers and had to try to avoid making any postural correction. Only when subjects stood still were they allowed to continue hopping. The time needed to complete the test and VAS scores for the perceived difficulty were assessed.In unstable ankles, ICCs of time values were >0.90 (SEM = 2.3 seconds); Spearman rho values of VAS scores were >0.80. When hopping on their unstable ankles, patients (41.1 ± 12.6 seconds) needed significantly more time to complete the test than healthy subjects (31.4 ± 5.0 seconds; test: P = 0.000; retest: P = 0.002) or when compared with their unaffected contralateral ankles (38.0 ± 7.1 seconds; test: P = 0.047; retest: P = 0.009). Only with respect to the dominant ankles, patients (median = 64 mm) perceived the test as significantly more difficult than did healthy subjects (median = 37 mm; test: P = 0.018; retest: P = 0.002). VAS scores of unstable ankles in patients (median = 50 mm) were significantly higher than their contralateral, unaffected ankles (median = 30 mm; test: P = 0.001; retest: P = 0.002).The multiple hop test is a reliable test demonstrating functional performance deficits in patients with CAI.