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Temporal variations of thrombolysis delivery and their influence on outcome have been reported with controversial results. In this large cohort study, we evaluated whether thrombolytic treatment has a within-day and weekly variability corresponding to circadian and weekly patterns of ischemic stroke onset, and whether these have impact on clinical outcome.We retrospectively analyzed patients with acute ischemic stroke receiving intravenous alteplase, prospectively included in the Safe Implementation of Treatments in Stroke-International Stroke Thrombolysis Register. Patients were grouped by treatment on day hours (08:00–19:59) or night hours (20:00–07:59) and treatment on weekdays and weekends. For each subgroup, we analyzed frequency of thrombolytic treatments, time intervals, and outcomes (3-month modified Rankin Scale score 0–2 as good functional outcome, mortality, symptomatic intracerebral hemorrhage).We included 21 513 patients. Considering the mean expected number of patients treated per hour (0.4) and per day of the week (9.8), if no temporal variations were present, patients were significantly treated more during day hours and weekdays (P<0.0001). Median door-to-needle and onset-to-treatment times were longer for patients treated during night hours and on weekends (P<0.01). After adjustment for confounding variables, treatment during day hours was an independent predictor of good functional outcome (odds ratio, 1.12; 95% confidence interval, 1.04–1.21; P=0.004), and patients treated during weekdays were at risk of higher mortality (odds ratio, 1.15; 95% confidence interval, 1.04–1.28; P=0.008).Frequency of thrombolytic treatment seems to follow the same circadian pattern of stroke incidence, whereas its correspondence to a weekly pattern is less clear. Time of treatment is an independent predictor of outcome.