Purpose: This study aimed to compare the relative exercise intensity at which the onset of accelerated muscle deoxygenation occurs during the 20-m shuttle run test (20mSRT) between boys and men and to examine whether the timing of the onset of acceleration appearance is related to 20mSRT performance in boys. Methods: Twenty-four boys performed the 20mSRT, during which concentration changes in oxygenated and deoxygenated hemoglobin and myoglobin (ΔOxy-Hb and ΔDeoxy-Hb, respectively) in the m. vastus lateralis were monitored using a portable near-infrared spectroscopy device. The boys’ data were compared with those of 29 men in a previous study. Results: An onset of accelerated decrease in Δ[Oxy-Hb − Deoxy-Hb] was found in 11 of the 24 boys (45.8%) and 20 of the 29 men (69.0%) and was found at a higher relative exercise intensity in the boys than in the men. The number of laps at which the onset of acceleration occurred correlated with total laps in the boys (r = .87). Conclusions: These findings demonstrate that the onset of accelerated muscle deoxygenation during the 20mSRT occurs at a higher relative exercise intensity in boys than in men. Our findings also show that the timing of the onset of acceleration appearance is associated with 20mSRT performance in boys.