The purpose of this study was to examine whether and how cycle time duration affects energy expenditure and substrate utilization during whole-body vibration (WBV). Nine men performed 3 squatting exercises in execution frequency cycles of 6, 4, and 2 seconds to 90 degrees knee flexion with vibration (Vb+) (frequency was set at 30 Hz and the amplitude of vibration was 4 mm) and without vibration (Vb-) during 3 minutes, each with an additional load of 30% of the subject's body weight. A 2-way analysis of variance for VO2 revealed a significant vibration condition main effect (p < 0.001) and a cycle time duration effect (p < 0.001). When differences were analyzed by Fisher's LSD test, cycle time duration of 2 seconds was significantly different from 4 and 6 seconds, both in Vb+ and Vb-. Total energy expenditure (EE(tot)), carbohydrate oxidation rate (EE(cho)), and fat oxidation rate (EE(fat)) demonstrated a significant vibration condition main effect (EE(tot): p < 0.01; EE(cho): p < 0.001; EE(fat): p < 0.001) and cycle time duration main effect (EE(tot) and EE(cho): p < 0.001; EE(fat): p < 0.01). EE(tot), EE(cho), and EE(fat) post hoc comparisons indicated that values for the 2-second test significantly differed from 4 and 6 seconds when compared in the same vibration condition. VO2 and EE values were greater in Vb+ than in Vb- conditions with the same cycle time duration. Our study confirms that squatting at a greater frequency helps to maximize energy expenditure during exercise with or without vibration. Therefore, cycle time duration must be controlled when vibration exercise is prescribed.