Background The ageing process can be influenced by energy intake and different macronutrients within the diet. The soluble form of the a-Klotho gene (called S-Klotho) is widely considered as a powerful anti-ageing biomarker. Objective To analyze the association of energy, dietary energy density and macronutrient intake with S-Klotho plasma levels in middle-aged sedentary adults. Methods A total of 72 (52.8% women) middle-aged sedentary adults (53.7 +/- 5.2 years old) participated in the study. Energy and macronutrients intake (i.e. fat, carbohydrate and protein) were assessed using three non-consecutive 24-h recalls. S-Klotho plasma levels were measured in the Ethylenediaminetetraacetic acid (EDTA) plasma using a solid-phase sandwich enzyme-linked immunosorbent assay. Results No association was observed between energy, dietary energy density or macronutrient intake and S-Klotho plasma levels in men (all P >= 0.1). We found an inverse association between energy, protein and carbohydrate intake with S-Klotho plasma levels in women (all P <= 0.043), which disappeared after controlling for age, lean mass index and sedentary time. An inverse association was observed between dietary energy density and S-Klotho plasma levels in women after controlling for covariates (all P <= 0.05). Conclusion In summary, the present study showed an inverse association of dietary energy density with S-Klotho plasma levels in middle-aged women. In addition, our data suggest that the associations between energy and macronutrient intake could be highly dependent on lean mass and sedentary time.