Context: Hamstring inflexibility is typically treated using therapeutic massage, stretching, and soft tissue mobilization. An alternative intervention is deep oscillation therapy (DOT). Currently, there is a lack of evidence to support DOT’s effectiveness to improve flexibility. Objective: To explore the effectiveness of DOT to improve hamstring flexibility. Design: Randomized single-cohort design. Setting: Research laboratory. Participants: Twenty-nine healthy, physically active individuals (self-reported activity of a minimum 200 min/wk). Interventions: All participants received a single session of DOT with randomization of the participant’s leg for the intervention. The DOT intervention parameters included a 1∶1 mode and 70% to 80% dosage at various frequencies for 28 minutes. Hamstring flexibility was assessed using passive straight leg raise for hip flexion using a digital inclinometer. Patient-reported outcomes were evaluated using the Copenhagen Hip and Groin Outcome Score and the Global Rating of Change (GRoC). Main Outcome Measure: The independent variable was time (pre and post). The dependent variables included passive straight leg raise, the GRoC, and the participant’s perceptions of the intervention. Statistical analyses included a dependent t test and a Pearson correlation. Results: Participants reported no issues with sport, activities of daily living, or quality of life prior to beginning the intervention study on the Copenhagen Hip and Groin Outcome Score. Passive straight leg raise significantly improved post-DOT (95% confidence interval, 4.48°–7.85°, P < .001) with a mean difference of 6.17 ± 4.42° (pre-DOT = 75.43 ± 21.82° and post-DOT = 81.60 ± 23.17°). A significant moderate positive correlation was identified (r = .439, P = .02) among all participants between the GRoC and the mean change score of hamstring flexibility. Participants believed that the intervention improved their hamstring flexibility (5.41 ± 1.02 points) and was relaxing (6.21 ± 0.86). Conclusions: DOT is an effective intervention to increase hamstring flexibility.