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Intramyocellular lipid (IMCL) variations in older men are poorly explored. In young adults, IMCL can be influenced by both diet and exercise interventions; this flexibility is related to aerobic fitness. We evaluated in active older adults the influence of maximal aerobic capacity on short-term diet and exercise-induced variations in IMCL stores.Intramyocellular lipids were measured by 1H magnetic resonance spectroscopy (1H-MRS) after a 3-day fat depletion-replenishment diet (IMCLFDR) and immediately after a 2-h exercise at 50% VO2 max (IMCLFDR_EX). To further explore diet influence, the protocol was repeated after a high-fat diet (HF), with both pre- and post-exercise measurements (IMCLHF and IMCLHF_EX).In active older men (69.8 ± 5.2 years), IMCLFDR was lowered by exercise (IMCLFDR = 3.45 ± 1.52 vs. IMCLFDR_EX = 2.74 ± 1.15 mmol kg−1 wet weight, P < 0.05), and exercise-induced variations were correlated to the initial store (P < 0.05, r = −0.72). IMCLFDR was linked with aerobic fitness (P < 0.05, r = 0.76), when adjusted by fat mass. IMCLHF was lower than IMCLFDR (P < 0.05), decreased after exercise (P < 0.05) and varied also as a function of initial store (P < 0.05, r = −0.89), but without link with aerobic fitness. Finally, diet-induced IMCL store variations were positively linked to aerobic fitness (P < 0.05, r = 0.89).Variations of the IMCL stores in physically active older adults appear related to aerobic fitness, with similarly fast adaptation to short-term interventions combining diet and exercise as young active adults.