Drop foot is common post-stroke, elevating fall risks and mobility limitations. It is caused by weakness and lack of control of the tibialis anterior muscle (TA), for which various rehabilitation treatments are used. A reliable objective estimate of changes in TA muscle morphology and composition can enhance treatment optimization. We aimed to ascertain 3D freehand ultrasound (3DfUS) reliability in measuring TA muscle volume, length, and echo intensity in stroke patients and healthy controls and its validity by comparing these features across legs, between patients and controls, and between clinical subgroups (i.e. patients with and without ankle contracture, spastic muscle overactivity, and foot dorsiflexor paresis). We included 9 stroke patients and 9 healthy controls to define reliability and 26 stroke patients and 28 healthy controls to define validity. For reliability, data were collected and processed by 2 different operators and processors. For inter- and intra-rater reliability, intra-class correlation coefficient (ICC) and standard error of measurement (SEM) were used. For validity, Wilcoxon-Signed-Ranked and Mann-Whitney U tests were used for comparisons between groups and subgroups. All measurements showed good to excellent inter- and intra-rater reliability (ICC: 0.816 to 0.997, SEM: 0.5% to 7.8%). Comparison analyses revealed no differences in muscle features among legs, groups, or subgroups. While the 3DfUS is a reliable method to define TA morphology and composition, its clinical validity needs further investigation into factors influencing muscle property changes across various age groups and post-stroke time points. Stroke; Skeletal muscle morphology; muscle composition; 3D freehand ultrasonography, Anterior Tibial Muscle</p