Background: Nerve damage is consistently demonstrated after subepineural injection in animal studies, but not after purposeful injection in patients participating in clinical studies. There is a need to better visualise nerves in order to understand the structural changes that occur during subepineural injection. Methods: We scanned the brachial plexuses of three anaesthetised pigs using micro-ultrasound imaging (55–22 MHz probe), inserted 21 gauge block needles into the radial, median, and axillary nerves, and injected two 0.5 ml boluses of saline into nerves at a rate of 12 ml min−1. Our objectives were to measure the area and diameter of nerves and fascicles, and to describe changes in nerve anatomy, comparing our findings with histology. Results: Images were acquired at 42 sites across 18 nerves in three pigs and compared dimensions (geometric ratio; 95% confidence interval; P value). As expected, the nerve cross-sectional area was greater in the proximal brachial plexus compared with the mid-plexus (2.10; 1.07–4.11; P<0.001) and the distal plexus (2.64; 1.42–4.87; P<0.001). Nerve area expanded after 0.5 ml injection (2.13; 1.48–3.08; P<0.001). Using microultrasound, subepineural injection was characterised by nerve and fascicle rotation, uniform, or localised swelling and epineural rupture. Micro-ultrasound revealed a unique pattern suggestive of subperineural injection after a median nerve injection, and good face validity with histology. Histology showed epineural trauma and inflammation to the perineurium. Conclusion: We accurately identified fascicles and real-time structural changes to peripheral nerves using micro-ultrasound. This is the first study to visualise in vivo and in real-time the motion of nerves and fascicles in response to anaesthetic needle insertion and fluid injection.