Non-uniform displacement is a well-documented phenomenon of healthy tendons that has shown to be reduced among injured and aging populations. Non-uniformity is considered a biomarker of tendon health, yet immediate response to physical exercise is unknown. This study examined acute changes in Achilles tendon (AT) non-uniform displacement in response to high strain magnitude isometric plantarflexion exercise. The reliability of the method was also examined. Fourteen healthy participants (7 men, 7 women, mean ± SD age:​ 26.4 ± 4.8 years) performed unilateral isometric plantarflexion exercise at 90 % of maximal voluntary isometric contractions (MVIC) with 5 sets of 4 repetitions, each lasting 3 s. The contralateral leg served as control. AT displacement was measured during ramp contractions to a constant torque level (30 % of MVIC) before the exercise, between the loading sets, and six times during 72-h recovery period. AT nonuniformity (difference between maximum and minimum displacement) was analyzed from sagittal B-mode ultrasound videos using speckle tracking. Two-way repeated measures ANOVA was used to compare the values across different timepoints. Non-uniformity did not change in response to exercise and was 2.99 ± 1.52 mm before and 3.19 ± 1.42 mm immediately after exercise. The reliability of non-uniformity between trials within a single measurement session varied from moderate to excellent (ICC:​ 0.680–0.920). While the isometric high strain plantarflexion exercise did not acutely alter the non-uniform displacement of the AT in young healthy adults, strenuous exercises containing knee and ankle joint angle changes should be investigated to confirm adaptability of AT non-uniform displacement.