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Background: Using uniaxial accelerometry approach in measuring physical activity levels of individuals with Down syndrome (DS) might underestimate their energy expenditure due to the unique mediolateral walking pattern. Therefore, the purpose of this study was to examine and compare the relationship between two types of accelerometer outputs, uniaxial and triaxial, and energy expenditure in individuals with and without DS., Methods: Eighteen participants with DS and 19 participants without DS wore a GT3X+ accelerometer and a portable metabolic system in three different walking conditions., Results: Correlations between V ̇ O 2 and each of the two accelerometer outputs (uniaxial: r = 0.75, triaxial: r = 0.75) were not significantly different among individuals without DS (z = 0.14, P = 0.89); however, significant differences in the relationship between V ̇ O 2 and accelerometer outputs (uniaxial: r = 0.53, triaxial: r = 0.64) were observed among individuals with DS (z = -1.72, P < 0.046)., Conclusions: The findings suggest that when using accelerometers to measure physical activity levels for individuals with DS, triaxial outputs may better predict physical activity levels., (© 2020 MENCAP and International Association of the Scientific Study of Intellectual and Developmental Disabilities and John Wiley & Sons Ltd.)