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Matching healthcare staff resources to patient needs in the ICU is a key factor for quality of care. We aimed to assess the impact of the staffing-to-patient ratio and workload on ICU mortality.We performed a multicenter longitudinal study using routinely collected hospital data.Information pertaining to every patient in eight ICUs from four university hospitals from January to December 2013 was analyzed.A total of 5,718 inpatient stays were included.None.We used a shift-by-shift varying measure of the patient-to-caregiver ratio in combination with workload to establish their relationships with ICU mortality over time, excluding patients with decision to forego life-sustaining therapy. Using a multilevel Poisson regression, we quantified ICU mortality-relative risk, adjusted for patient turnover, severity, and staffing levels. The risk of death was increased by 3.5 (95% CI, 1.3–9.1) when the patient-to-nurse ratio was greater than 2.5, and it was increased by 2.0 (95% CI, 1.3–3.2) when the patient-to-physician ratio exceeded 14. The highest ratios occurred more frequently during the weekend for nurse staffing and during the night for physicians (p < 0.001). High patient turnover (adjusted relative risk, 5.6 [2.0–15.0]) and the volume of life-sustaining procedures performed by staff (adjusted relative risk, 5.9 [4.3–7.9]) were also associated with increased mortality.This study proposes evidence-based thresholds for patient-to-caregiver ratios, above which patient safety may be endangered in the ICU. Real-time monitoring of staffing levels and workload is feasible for adjusting caregivers’ resources to patients’ needs.