Muscle volume and length are important parameters for examining the force-generating capabilities of muscle and their evaluation is necessary in studies that investigate muscle morphology and mechanical changes due to age, function, pathology, surgery and training. In this study, we assessed the validity and reliability of in vivo muscle volume and muscle belly length measurement using a multiple sweeps freehand 3D ultrasound (3DUS). The medial gastrocnemius of 10 subjects was scanned at three ankle joint angles (15 degrees , 0 degrees and -15 degrees dorsiflexion) three times using the freehand 3DUS and once on the following day using magnetic resonance imaging (MRI). All freehand 3DUS and MRI images were segmented, volumes rendered and volumes and muscle belly lengths measured. The freehand 3DUS overestimated muscle volume by 1.9+/-9.1 mL, 1.1+/-3.8% difference and underestimated muscle belly length by 3.0+/-5.4mm, 1.3+/-2.2% difference. The intra-class correlation coefficients (ICC) for repeated freehand 3DUS system measures of muscle volume and muscle belly length were greater than 0.99 and 0.98, respectively. The ICCs for the segmentation process reliability for the freehand 3DUS system and MRI for muscle volume were both greater than 0.99 and muscle belly length were 0.97 and 0.99, respectively. Freehand 3DUS is a valid and reliable method for the measurement of human muscle volume and muscle belly length in vivo. It could be used as an alternative to MRI for measuring in vivo muscle morphology and thus allowing the determination of PCSA and estimation of the force-generating capacity of individual muscles within the setting of a biomechanics laboratory.