PURPOSE: To examine the repeatability of detecting pupillary dilation lag in patients with Horner syndrome. DESIGN: Retrospective interventional study. METHODS: Setting: Single referral institution. Patient population: Fifteen patients with unilateral Horner syndrome and 16 subjects with physiologic anisocoria. Intervention procedure: Each subject underwent four pupillometric recordings in darkness. The asymmetry of pupillodilation between the two eyes was calculated as the change in anisocoria between five seconds and 15 seconds in darkness. Pupillary dilation lag was considered present if the asymmetry measured > or =0.4 mm. Main outcome measure: Asymmetry of pupillodilation over four determinations. RESULTS: All subjects demonstrated fluctuations in the calculated asymmetry of pupillodilation. Eight patients (53%) with Horner syndrome demonstrated dilation lag on the first determination; 13 patients (87%) eventually demonstrated it during four determinations. CONCLUSIONS: Pupillary dilation lag is intermittently present in most patients with Horner syndrome. Repeated observations improve the detection rate of dilation lag, a confirmatory sign of an oculosympathetic deficit. Its absence does not rule out Horner syndrome.