This cross-sectional study investigates the relationship between the shed form of the Klotho protein (S-Klotho) in plasma, and cardiometabolic risk in healthy, sedentary adults. The study subjects were 214 healthy, sedentary adults ((similar to)64% women). Data were collected during the baseline assessments of two randomized controlled trials: The FIT-AGEING study (n=74 [(similar to)50% women] middle-aged adults aged 40-65 years) and the ACTIBATE study (n=140 [(similar to)70% women] young adults aged 18-25 years). A sex-specific cardiometabolic risk score was calculated for each subject based on waist circumference, systolic and diastolic blood pressure, and plasma glucose, high-density lipoprotein cholesterol and triglycerides. A significant inverse relationship was detected between S-Klotho and the cardiometabolic risk score of both the middle-aged men and women (beta=-0.658, R-2=0.433, P<0.001 and beta=-0.442, R-2=0.195, P=0.007) which persisted after adjusting for actual age, energy intake, and VO(2)max. No significant association was found between S-Klotho and cardiometabolic risk score for the young, healthy adults (P>0.5), nor for the young, healthy men and women when analysed separately (all P>0.1). In conclusion, in healthy, sedentary, middle-aged adults, but not in young, healthy, sedentary adults, higher plasma S-Klotho concentrations are associated with a lower cardiometabolic risk score.