Knee osteoarthritis (KOA) can cause functional disability. Neuromuscular function is relevant in the development and progression of KOA. It can be evaluated by the analysis of the surface electromyography (sEMG), which has an important role in the understanding of KOA. Whole-body vibration (WBV) is an intervention suggested to treat KOA. The objective of this work was to verify the effectiveness of WBV on the functionality of lower limbs by the electromyographic profile of the vastus lateralis (VL) muscles during the five-repetition chair stand test (5CST) in patients with KOA. This was a two-period crossover trial study (8-week washout). Nineteen patients with KOA were allocated to the group submitted to WBV (WBVG), with peak-to-peak displacement of 2.5 to 7.5 mm, frequency from 5 to 14 Hz, and acceleration peak from 0.12 to 2.95 g, or to the control group (0 Hz) (2 days per week for 5 weeks). The 5CST and the sEMG of the VL during 5CST were evaluated before and after the interventions. Results: Significant differences in 5CST were evident only in WBVG (p = 0.018), showing a decrease of the execution time. The sEMG profile showed no significative difference. Therefore, only 10 sessions of WBV with comfortable posture can bring about improvement in functionality of KOA patients without alteration of the muscle excitation.