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Abstract Background Muscle wasting in critically ill patients is associated with negative clinical outcomes. Ultrasound quadriceps femoris muscle assessment may constitute a convenient tool to evaluate muscle wasting. Nevertheless, its reliability remains uncertain. Our primary aim was to study the intra- and inter-observer reliability of this technique. Our secondary aim was to assess the evolution of the quadriceps muscle during the first 3 weeks after ICU admission and its possible association with nutritional intake. Methods This observational study included patients expected to stay more than 7 days in the ICU. Ultrasound quadriceps muscle thickness was measured with a 12 MHz linear transducer, by two trained physicians, on D1, D3, D5, D7 and D21. Two measurements sites were evaluated: on the midpoint or on the two-thirds of the length between the anterior superior iliac spine and the upper border of the patella. Intra and inter-observer reliability was assessed by calculating the intra-class correlation coefficient (ICC). Results A total of 280 ultrasound quadriceps thickness measurements were performed on 29 critically ill patients. Intra-observer reliability’s ICC was 0.74 [95% CI 0.63; 0.84] at the “midpoint” site and 0.83 [95% CI 0.75; 0.9] at the “two-thirds” site. Inter-observer reliability’s ICC was 0.76 [95% CI, 0.66; 0.86] at the “midpoint” site and 0.81 [95% CI, 0.7; 0.9] at the “two-thirds” site. Quadriceps femoris muscle thickness decreased over 16% within the first week after ICU admission. No correlation was found between muscle loss and caloric (p = 0.96) or protein (p = 0.80) debt over the first week. Conclusion The assessment by ultrasonography of the quadriceps muscle thickness reveals good intra- and inter-observer reliability and may constitute a promising tool to evaluate the effect of nutritional-based interventions on muscle wasting in critically ill patients. Trial registration “Committee for the Protection of Human Subjects in Biomedical Research” - Paris Ile de France VI Pitié-Salpêtrière – 10/07/2014. French Data Protection Committee (“Commission Nationale Informatique et Libertés”) - #1771144.