Context: The mechanisms that contribute to the development of chronic ankle instability are not understood. Investigators have developed a hypothetical model in which neuromuscular alterations that stem from damaged ankle ligaments are thought to affect periarticular and proximal muscle activity. However, the retrospective nature of these studies does not allow a causal link to be established. Objective: To assess temporal alterations in the activity of 2 periarticular muscles of the rat ankle and 2 proximal muscles of the rat hind limb after an ankle sprain. Design: Controlled laboratory study. Setting: Laboratory. Patients or Other Participants: Five healthy adult male Long Evans rats (age = 16 weeks, mass = 400.0 ± 13.5 g). Intervention(s): Indwelling fine-wire electromyography (EMG) electrodes were implanted surgically into the biceps femoris, medial gastrocnemius, vastus lateralis, and tibialis anterior muscles of the rats. We recorded baseline EMG measurements while the rats walked on a motor-driven treadmill and then induced a closed lateral ankle sprain by overextending the lateral ankle ligaments. After ankle sprain, the rats were placed on the treadmill every 24 hours for 7 days, and we recorded postsprain EMG data. Main Outcome Measure(s): Onset time of muscle activity, phase duration, sample entropy, and minimal detectable change (MDC) were assessed and compared with baseline using 2-tailed dependent t tests. Results: Compared with baseline, delayed onset time of muscle activity was exhibited in the biceps femoris (baseline = −16.7 ± 54.0 milliseconds [ms]) on day 0 (5.2 ± 64.1 ms; t4 = −4.655, P = .043) and tibialis anterior (baseline = 307.0 ± 64.2 ms) muscles on day 3 (362.5 ± 55.9 ms; t4 = −5.427, P = .03) and day 6 (357.3 ± 39.6 ms; t4 = −3.802, P = .02). Longer phase durations were observed for the vastus lateralis (baseline = 321.9 ± 92.6 ms) on day 3 (401.3 ± 101.2 ms; t3 = −4.001, P = .03), day 4 (404.1 ± 93.0 ms; t3 = −3.320, P = .048), and day 5 (364.6 ± 105.2 ms; t3 = −3.963, P = .03) and for the tibialis anterior (baseline = 103.9 ± 16.4 ms) on day 4 (154.9 ± 7.8 ms; t3 = −4.331, P = .050) and day 6 (141.9 ± 16.2 ms; t3 = −3.441, P = .03). After sprain, greater sample entropy was found for the vastus lateralis (baseline = 0.7 ± 0.3) on day 6 (0.9 ± 0.4; t4 = −3.481, P = .03) and day 7 (0.9 ± 0.3; t4 = −2.637, P = .050) and for the tibialis anterior (baseline = 0.6 ± 0.4) on day 4 (0.9 ± 0.5; t4 = −3.224, P = .03). The MDC analysis revealed increased sample entropy values for the vastus lateralis and tibialis anterior. Conclusions: Manually inducing an ankle sprain in a rat by overextending the lateral ankle ligaments altered the complexity of muscle-activation patterns, and the alterations exceeded the MDC of the baseline data.