S-klotho, the shed form of α-klotho, is thought to be an ageing suppressor with functions related to the physiology of energy metabolism. However, it remains unknown whether ageing biomarkers such as S-klotho and/or chronological ageing are associated in any way with basal metabolic rate (BMR) and fuel oxidation in basal conditions and during exercise. The present work investigates the association of BMR and fuel oxidation in basal conditions and during exercise, with plasma S-klotho in middle-aged, sedentary adults. BMR was measured by indirect calorimetry in 74 such subjects (53% women; age 53.7±5.1 years) following standard procedures, and their fuel oxidation estimated via stoichiometric equations. The maximal fat oxidation during exercise (MFO) and the intensity of exercise that elicits MFO (Fatmax) were determined using a walking graded exercise test. No relationship was seen between BMR and plasma S-klotho (P>0.1), although both basal fat oxidation and MFO showed positive associations with this protein (both P<0.001); these relationships persisted after controlling for age, sex and fat mass. However, no significant associations were seen between BMR, basal fat oxidation or MFO and chronological age (all P>0.1). The present findings suggest that basal fat oxidation and MFO are strongly associated with plasma S-klotho in middle-aged sedentary adults. These results support the idea that metabolic flexibility is a powerful predictor of biological ageing.