Background: Water treadmills are frequently used in the rehabilitation of dogs, for example with the purpose of re-building muscular strength after surgery. However, little is known about how different water depths and velocities affect the muscular workload during aquatic locomotion. This study used acoustic myography to assess hind limb muscle fiber activation in 25 healthy large-breed dogs walking in a water treadmill. Acoustic myography sensors were attached to the skin over the vastus lateralis of the quadriceps and the biceps femoris muscles. The dogs walked at two velocities (30 and 50 m/min) and four water depths: bottom of the pads, hock, stifle and mid-femur. Acoustic myograph signals were analyzed for changes in three muscle function parameters: efficiency/coordination (E-score) and spatial (S-score) and temporal (T-score) summation. Results: Differences between E, S, and T were statistically significant compared across different speeds (30, 50) and water levels (0, 1, 2, 3) using a one-way ANOVA with multiple comparisons (Tukey; Geisser-Greenhouse correction) as well as a two-tailed one sample t-test. At 50 m/min in water at the mid-femur, the biceps femoris was less efficient (P < 0.001) and recruited more fibers (P = 0.01) at a higher firing rate (P = 0.03) compared to working in shallower water, while the vastus lateralis was also less efficient (P < 0.01), but spatial and temporal summation did not change significantly. At 30 m/min, biceps efficiency was reduced (P < 0.01) when water was at the mid-femur compared to the bottom of the pads level. Walking in stifle- or hock-deep water did not show increased muscle activation for either muscle compared to walking in water at the bottom of the pads. Conclusion: More muscle activation was required to walk in water at a depth at the level of the mid-femur compared to shallower water, and this exercise was more demanding for the biceps femoris, a muscle engaged in propulsion, than for vastus lateralis. These findings may help practitioners towards making more precise rehabilitation protocols.