The aim of this study was to examine associations of ectopic adipose tissue (AT) with skeletal muscle (SM) mitochondrial bioenergetics in older adults.

Cross‐sectional data from 829 adults ≥70 years of age were used. Abdominal, subcutaneous, and visceral AT and thigh muscle fat infiltration (MFI) were quantified by magnetic resonance imaging. SM mitochondrial energetics were characterized in vivo (³¹P‐magnetic resonance spectroscopy; ATP_max) and ex vivo (high‐resolution respirometry maximal oxidative phosphorylation [OXPHOS]). ActivPal was used to measure physical activity ([PA]; step count). Linear regression adjusted for covariates was applied, with sequential adjustment for BMI and PA.

Independent of BMI, total abdominal AT (standardized [Std.] β = −0.21; R² = 0.09) and visceral AT (Std. β = −0.16; R² = 0.09) were associated with ATP_max (p < 0.01; n = 770) but not following adjustment for PA (p ≥ 0.05; n = 658). Visceral AT (Std. β = −0.16; R² = 0.25) and thigh MFI (Std. β = −0.11; R² = 0.24) were associated with carbohydrate‐supported maximal OXPHOS independent of BMI and PA (p < 0.05; n = 609). Total abdominal AT (Std. β = −0.19; R² = 0.24) and visceral AT (Std. β = −0.17; R² = 0.24) were associated with fatty acid‐supported maximal OXPHOS independent of BMI and PA (p < 0.05; n = 447).

Skeletal MFI and abdominal visceral, but not subcutaneous, AT are inversely associated with SM mitochondrial bioenergetics in older adults independent of BMI. Associations between ectopic AT and in vivo mitochondrial bioenergetics are attenuated by PA.