This study aimed to investigate the effects of different training modalities on the soluble Klotho (S-Klotho) plasma levels in sedentary middle-aged adults. A total of 74 middle-aged adults (53.4 +/- 5.0 years old; 52.7% women) were enrolled in the FIT-AGEING study. We conducted a 12-week randomised controlled trial. The participants were randomly assigned to 4 different groups: (i) a control group (no exercise), (ii) a physical activity recommendation from the World Health Organization group (PAR), (iii) a high intensity interval training group (HIIT), and (iv) a high intensity interval training group adding whole-body electromyostimulation training group (HIIT-EMS). S-Klotho plasma levels, anthropometric measurements, and body composition variables were measured before and after the intervention programme. All exercise training modalities induced an increase in the S-Klotho plasma levels (all P <= 0.019) without statistical differences between them (all P >= 0.696). We found a positive association between changes in lean mass index and changes in the S-Klotho plasma levels, whereas a negative association was reported between changes in fat mass outcomes and changes in the S-Klotho plasma levels after our intervention study. In conclusion, our results suggest that the link between exercise training and the increase in S-Klotho plasma levels could be mediated by a decrease of fat mass and an increase of lean mass.