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Open-access scripts to perform muscle anatomical cross-sectional area (ACSA) evaluation in ultrasound images are currently unavailable. This study presents a novel semi-automatic ImageJ script (named "ACSAuto") for quantifying the ACSA of lower limb muscles. We compared manual ACSA measurements from 180 ultrasound scans of vastus lateralis (VL) and rectus femoris (RF) muscles to measurements assessed by the ACSAuto script. We investigated inter- and intra-investigator reliability of the script. Consecutive-pairwise intra-class correlations (ICC) and standard error of measurement (SEM) with 95% compatibility interval were calculated. Bland-Altman analyses were employed to test the agreement between measurements. Comparing manual and ACSAuto measurements, ICCs and SEMs ranged from 0.96 to 0.999 and 0.12 to 0.96 cm 2 (1.2-5.9%) and mean bias was smaller than 0.5 cm 2 (4.3%). Inter-investigator comparison revealed ICCs, SEMs and mean bias ranging from 0.85 to 0.999, 0.07 to 1.16 cm 2 (0.9-7.6%) and - 0.16 to 0.66 cm 2 (- 0.6 to 3.2%). Intra-investigator comparison revealed ICCs, SEMs and mean bias between 0.883-0.998, 0.07-0.93 cm 2 (1.1-7.6%) and - 0.80 to 0.15 cm 2 (- 3.4 to 1.8%). Image quality needs to be high for efficient and accurate ACSAuto analyses. Taken together, the ACSAuto script represents a reliable tool to measure RF and VL ACSA, is comparable to manual analysis and can reduce time needed to evaluate ultrasound images.