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This study examined the effects of cycling training on mechanomyographic median power frequency (MMG_MDF)–torque relationships of the vastus lateralis (VL). Ten males (Age ± SD; 20.20 ± 1.87 years) and 14 females (21.93 ± 5.33 years) performed isometric trapezoidal muscle actions with the knee extensors at 40% maximal voluntary contraction (MVC) before (PRE) and following 10 weeks of cycling training at the same absolute submaximal torque as pre-training (POST_ABS). MMG_MDF–torque relationships (increasing and decreasing segment) were log-transformed and <i>b</i> terms (slopes) were calculated. MMG_MDF was averaged during steady torque. For POST_ABS, the <i>b</i> terms for the females (0.133 ± 0.190) were greater than for the males (−0.083 ± 0.200; <i>p</i> = 0.013) and compared to PRE (0.008 ± 0.161; <i>p</i> = 0.036). At PRE, the <i>b</i> terms for the linearly increasing-muscle action (0.123 ± 0.192) were greater compared to the linearly decreasing-muscle action (−0.061 ± 0.188; <i>p</i> < 0.001), whereas no differences existed between muscle actions for POST_ABS (<i>p</i> > 0.05). In conclusion, 10 weeks of cycling training resulted in different motor unit (MU) control strategies between sexes and altered MU control strategies between muscle actions for the VL during a moderate-intensity contraction.