The ability to assess welfare is an important refinement that will ensure the good condition of animals used in experimentation. The present study investigated the impact of invasive procedures on the patterns of movement of zebrafish (Danio rerio). Recordings were made before and after fin clipping, PIT tagging and a standard pain test and these were compared with control and sham handled zebrafish. The fractal dimension (FD) from the 3D trajectories was calculated to determine the effect of these treatments on the complexity of movement patterns. While the FD of zebrafish trajectories did not differ over time in either the control or sham group, the FDs of the treatment groups reduced in complexity. The FD of fish injected with different strengths of acetic acid declined in a dose-dependent manner allowing us to develop an arbitrary scale of severity of the treatments. The 3D trajectory plots from some groups indicated the presence of repetitive swimming patterns akin to stereotypical movements. When administered with lidocaine, which has analgesic properties, the movement complexity of fin clipped fish reverted to a pattern that resembled that of control fish. Fractal analysis of zebrafish locomotion could potentially be adopted as a tool for fish welfare assessment.