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The objective of this study was to evaluate the prognostic value of lactate levels during the first 12 hours after shock development and to identify the optimal timing for repeated lactate measurements in patients with septic shock.We conducted a retrospective cohort study using a prospective data registry, and enrolled 2,226 consecutive adult patients with septic shock between January 2010 and December 2015. Blood lactate was measured at shock development, and after 2, 4, 6 and 12 hours (T0, T2, T4, T6 and T12) during protocol-driven resuscitation bundle therapy. The prognostic value of lactate levels for 28-day mortality was analyzed using logistic regression and receiver operating characteristic curve analysis.A total of 829 patients with septic shock were included in the study, among whom 211 died during the study period. The lactate levels at each time point were associated with increased 28-day mortality, and the lactate level at 6 hours had the greatest prognostic value (area under the curve of T0 = 0.61; T2 = 0.65; T4 = 0.69; T6 = 0.72 and T12 = 0.62, and odds ratio (OR) of T0, 1.17 [95% CI: 1.11-1.23]; T2, 1.23 [95% CI: 1.17-1.30]; T4, 1.30 [95% CI: 1.22-1.38]; T6, 1.33 [95% CI: 1.26-1.42] and T12, 1.24 [95% CI: 1.19-1.30]). Hyperlactatemia over 2 mmol/L and 4 mmol/L at 6 hours from shock was associated with 4-times higher mortality (≥2 mmol/L, OR = 3.89 [95% CI: 2.48-6.09]; ≥4 mmol/L, OR = 3.93 [95% CI: 2.83-5.44]).During the first 12 hours following shock development, the optimal time point of repeated blood lactate measurement was 6 hours, which was the greatest prognostic value for mortality.