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The iliotibialis lateralis pars postacetabularis (ILPO) is the largest muscle in the hindlimb of the guinea fowl and is thought to play an important role during the stance phase of running, both absorbing and producing work. Using sonomicrometry and electromyography, we examined whether the ILPO experiences differential strain between proximal, central and distal portions of the posterior fascicles. When the ILPO is being lengthened while active, the distal portion was found to lengthen significantly more than either the proximal or central portions of the muscle. Our data support the hypothesis that the distal segment lengthened farther and faster because it began activity at shorter sarcomere lengths on the ascending limb of the length–tension curve. Probably because of the self-stabilizing effects of operating on the ascending limb of the length–tension curve, all segments reached the end of lengthening and started shortening at the same sarcomere length. During shortening, this similarity in sarcomere length among the segments was maintained, as predicted from force–velocity effects, and shortening strain was similar in all segments. The differential active strain during active lengthening is thus ultimately determined by differences in strain during the passive portion of the cycle. The sarcomere lengths of all segments of the fascicles were similar at the end of active shortening, but after the passive portion of the cycle the distal segment was shorter. Differential strain in the segments during the passive portion of the cycle may be caused by differential joint excursions at the knee and hip acting on the ends of the muscle and being transmitted differentially by the passive visco-elastic properties of the muscle. Alternatively, the differential passive strain could be due to the action of active or passive muscles in the thigh that transmit force to the IPLO in shear. Based on basic sarcomere dynamics we predict that differential strain is more likely to occur in muscles undergoing active lengthening at the beginning of contraction than those undergoing only shortening. * ILPO : iliotibialis lateralis pars postacetabularis L : the length at the middle of the plateau region of the length–tension curve (optimal length) L max,st : maximum length of the ILPO occurring during stance L max,sw : maximum length of the ILPO occurring during swing L min,fd : minimum length of the ILPO that occurred near the time of foot-down L min,to : minimum length of the ILPO that occurred near the time of toe-off L off : length of the ILPO 25 ms after the stop of the EMG activity L on : length of the ILPO 25 ms after the start of the EMG activity L sc : in vivo sarcomere lengths L sc,ref : reference sarcomere length L so : in vivo segment lengths measured by sonomicrometry L so,ref : sonomicrometer length t al : duration of active lengthening V al : average velocity during active lengthening V as : velocity during active shortening ΔL : passive shortening Δ L al : active lengthening Δ L as : active shortening Δ L as : active shortening Δ L pl : passive lengthening