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Many studies examine sarcomere dynamics in single fibres or length–tension dynamics in whole muscles in vivo or in vitro, but few studies link the various levels of organisation. To relate data addressing in vitro muscle segment behaviour with in vivo whole muscle behaviour during locomotion, we measured in vivo strain patterns of muscle segments using three sonomicrometry crystals implanted along a fascicle of the semimembranosus muscle in the American toad (Bufo americanus; n= 6) during hopping. The centre crystal emitted an ultrasonic signal, while the outer crystals received the signal allowing the instantaneous measurement of lengths from two adjacent muscle segments. On the first day, we recorded from the central and distal segments. On the second day of recordings, the most distal crystal was moved to a proximal position to record from a proximal segment and the same central segment. When the toads hopped a distance of two body lengths, the proximal and central segments strained −15.1 ± 6.1 and −14.0 ± 4.9 % (i.e. shortening), respectively. Strain of the distal segment, however, was significantly lower and more variable in pattern, often lengthening before shortening during a hop. From rest length, the distal segment initially lengthened by 2.6 ± 2.0 % before shortening by 6.5 ± 3.2 % at the same hop distance. Under in vitro conditions, the central segment always shortened more than the distal segment, except when passively cycled, during which the segments strained similarly. When the whole muscle was cycled sinusoidally and stimulated phasically in vitro, the two adjacent segments strained in opposite directions over much (up to 34 %) of the cycle. These differences in strain amplitude and direction imply that two adjacent segments can not only produce and/or absorb varying amounts of mechanical energy, but can also operate on different regions of their force–length and force–velocity relationships when activated by the same neural signal. Understanding regional differences in contractile dynamics within muscles is therefore important to linking our understanding of sarcomere behaviour with whole muscle behaviour during locomotion.