ObjectiveNon-invasive identification of motoneuron (MN) activity is commonly done using (EMG). However, surface EMG (sEMG) signals detect only superficial sources, at less than approximately 10-mm depth. Intramuscular EMG can detect deep sources, but it is limited to sources within a few mm of the detection site. Conversely, ultrasound (US) images have high spatial resolution across the whole muscle cross-section. The activity of MNs can be extracted from US images due to the movements that MN activation generates in the innervated muscle fibers. Current US-based decomposition methods can accurately identify the location and average twitch induced by MN activity. However, they cannot accurately detect MN discharge times.MethodsHere, we present a method based on the convolutive blind source separation of US images to estimate MN discharge times with high accuracy. The method was validated across 10 participants using concomitant sEMG decomposition as the ground truth.Results140 unique MN spike trains were identified from US images, with a rate of agreement (RoA) with sEMG decomposition of 87.4 ± 10.3 %. Over 50% of these MN spike trains had a RoA greater than 90%. Furthermore, with US, we identified additional MUs well beyond the sEMG detection volume, at up to >30 mm below the skin.ConclusionThe proposed method can identify discharges of MNs innervating muscle fibers in a large range of depths within the muscle from US images.SignificanceThe proposed methodology can non-invasively interface with the outer layers of the central nervous system innervating muscles across the full cross-section.