Background: Stroke survivors with impaired control of the ankle due to stiff plantarflexors often experience abnormal posture control, which affects balance and locomotion. Forceful stretching may decrease ankle stiffness and improve balance. Recently, a robot-aided stretching device was developed to decrease ankle stiffness of patient post-stroke, however, their benefits compared to manual stretching exercises have not been done in a randomized controlled trial, and the correlations between the ankle joint biomechanical properties and balance are unclear. Objective: To compare the effects of robot-aided to manual ankle stretching training in stroke survivors with the spastic ankle on the ankle joint properties and balance function post-stroke, and further explore the correlations between the ankle stiffness and balance. Methods: Twenty inpatients post-stroke with ankle spasticity received 20 minutes of stretching training daily over two weeks. The experimental group used a robot-aided stretching device, and the control group received manual stretching. Outcome measures were evaluated before and after training. The primary outcome measure was ankle stiffness. The secondary outcome measures were passive dorsiflexion ranges of motion, dorsiflexor muscle strength, Modified Ashworth Scale (MAS), Fugl-Meyer Motor Assessment of Lower Extremity (FMA-LE), Berg Balance Scale (BBS), Modified Barthel Index (MBI), and the Pro-Kin balance test. Results: After training, two groups showed significantly within-group improvements in dorsiflexor muscle strength, FMA-LE, BBS, MBI (P < 0.05). The between-group comparison showed no significant differences in all outcome measures (P > 0.0025). The experimental group significantly improved in the stiffness and passive range of motion of dorsiflexion, MAS. In the Pro-Kin test, the experimental group improved significantly with eyes closed and open (P < 0.05), but significant improvements were found in the control group only with eyes open (P < 0.05). Dorsiflexion stiffness was positively correlated with the Pro-Kin test results with eyes open and the MAS (P < 0.05). Conclusions: The robot-aided and manual ankle stretching training provided similar significant improvements in the ankle properties and balance post-stroke. However, only the robot-aided stretching training improved spasticity and stiffness of dorsiflexion significantly. Ankle dorsiflexion stiffness was correlated with balance function. Clinical Trial Registration: www.chictr.org.cn ChiCTR2000030108.

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