Restless legs syndrome (RLS) is very common in hemodialysis patients. RLS induces motor excitability and discomfort during rest periods, and those symptoms have also been observed during hemodialysis sessions. The aim of the study was to assess whether a single bout of exercise could reduce periodic limb movements (PLM) occurring during hemodialysis. Eighteen hemodialysis patients were eligible and participated in the study. Using the RLS criteria and further verified by the presence of PLM during sleep, patients were divided to non-RLS and RLS groups. Three scenarios were studied during three different sessions: 1) light exercise, including cycling for 45 minutes with no added resistance, 2) heavy exercise, including cycling for 45 minutes with a resistance set at 60% of their exercise capacity, and 3) no exercise, including rest for the same period of time. In all sessions, PLM per hour of hemodialysis (PLM/hHD) was recorded. A single bout of either light or heavy exercise was equally effective in significantly reducing PLM/hHD in patients with RLS compared with the no-exercise scenario, whereas in non-RLS patients, no effect was observed. Independent of intensity, a single bout of intradialytic exercise reduces PLM/hHD in hemodialysis patients with RLS. Further research is needed to establish the acute role of exercise in ameliorating the RLS symptoms.