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Background: Randomized controlled trials have demonstrated that a restrictive red blood cell (RBC) transfusion strategy lowers transfusion frequency without affecting mortality. However, the external validity of these trials has not been tested in a large cohort. The purpose was to estimate the effect of introducing a National Clinical Guideline (NCG) for a restrictive hemoglobin transfusion threshold on transfusion frequency and mortality in hip fracture patients > 65 years old. Methods: A consecutive cohort study of hip fracture patients > 65 years old residing in the southern region of Denmark was conducted using prospectively gathered data from registers during two separate 1-year time periods. The first period from October 1, 2012, to September 30, 2013, included 1494 patients and used a liberal transfusion threshold, whereas the second period from October 1, 2015, to September 30, 2016, including 1414 participants used a restrictive threshold from the NCG. Participant data for age, sex, body mass index, Charlson Comorbidity Index, time to surgery, and death were retrieved from the Danish Interdisciplinary Registry of Hip Fractures and were merged with RBC transfusion and medication data extracted from the Danish Transfusion and Odense Pharmacoepidemiological Databases, respectively. Cox proportional hazards models were used to test relative mortality risk for the restrictive group compared with the liberal group at 30 and 90 days. Results: Overall RBC transfusions decreased from 42 to 30% (p < 0.001). The 30-day mortality rate (95% CI) was 9% (8;11) in the restrictive group and 13% (11;14) in the liberal group (p < 0.008), whereas the adjusted relative mortality risk was 0.72 (0.57;0.91). The 90-day mortality rate was 15% (13;17) in the restrictive group and 19% (17;21) in the liberal group, whereas the adjusted relative mortality risk was 0.78 (0.65;0.94). Conclusion: These data suggest that the introduction of an NCG on restrictive blood transfusion leads to lower transfusion frequency in hip fracture patients > 65 years old. Even though this reduction is associated with decreased mortality at both 30 and 90 days, it may be explained by other issues than restrictive transfusion strategy. There has been an improvement in the mortality of hip fracture patients in Denmark, and we suggest that a restrictive transfusion strategy does not lead to increased mortality. [ABSTRACT FROM AUTHOR]