Abstract The Achilles tendon (AT) is composed of three distinct subtendons, each arising from one of the three heads of the triceps surae muscles: gastrocnemius medialis (GM), gastrocnemius lateralis (GL), and soleus (SOL). These subtendons exhibit a twisted structure, classified as low (Type I), medium (Type II), and high (Type III) twist, based on cadaveric studies. Nevertheless, the in vivo investigation of AT twist is notably scarce, resulting in a limited understanding of its functional significance. The aim of this study was to give insights into the complex 3D AT structure in vivo. A total of 30 healthy participants underwent individual stimulation of each of the triceps surae muscles at rest with the foot attached to the pedal of an isokinetic dynamometer. Ultrasound images were captured to concomitantly examine the displacement of the superficial, middle and deep AT layers. SOL stimulation resulted in the highest AT displacement followed by GM and GL stimulation. Independent of the muscle stimulated, non‐uniformity within the AT was observed with the deep layer exhibiting more displacement compared to the middle and superficial layers, hence important inter‐individual differences in AT displacement were noticeable. By comparing these individual displacement patterns during targeted stimulations with insights from cadaveric twist classifications on each subtendon area, our classification identified 19 subjects with a ‘low’ twist and 11 subjects with a ‘high’ twist. These findings enable us to move beyond cadaveric studies and relate the twisted microstructure of the AT in vivo to its dynamic behaviour.