This application is an ancillary study of the PREDIMED-Plus trial to examine the effects of a diet and lifestyle intervention on incidence of T2D as a secondary endpoint among 4,770 participants who were non-diabetics at baseline. PREDIMED-Plus is an ongoing primary cardiovascular prevention trial (www.predimedplus.com), which has recruited 6,874 women and men (aged 55 to 75 years) between October 2013 and December 2016. Participants were randomly allocated to 2 groups: an energy-reduced Mediterranean diet (MedDiet) group, with physical activity and behavioral support for weight loss; or a control group receiving low-intensity advice on the MedDiet. The intervention will last 6 years. We will examine four Specific Aims. First, we will evaluate whether an intensive lifestyle intervention consisting of an energy-reduced MedDiet, increased physical activity, and weight loss, reduces the incidence of T2D comparied with the control group (intervention group n=2,384; control group n=2,451). Second, we will evaluate the effect of the lifestyle intervention on total fat mass and visceral adipose tissue (VAT) measured by Dual-energy X-ray Absorptiometry (DXA) after 1, 3, and 6-year intervention in a subsample (n=1,569). Third, we will examine whether the lifestyle intervention modifies the association of the metabolite risk score (MRS) with the incidence of T2D in a nested case-control study (n=620 incident T2D cases and 620 matched controls). These analyses will be replicated in the multi-ethnic US Diabetes Prevention Program (DPP, 775 cases and 1,386 non-cases from the placebo/lifestyle groups). Fourth, we will examine whether the intervention induces beneficial changes in stool metabolites from baseline to year 1, in a subsample of 250 participants in the intervention group and 250 in the control group. We will also examine whether changes in stool metabolites are associated with changes in gut microbiome induced by the intervention. The preliminary results among the PREDIMED-Plus participants showed that the intervention significantly reduced body weight and HbA1c, which was sustained over three years. This study builds upon a large RCT and will be the first and largest of its kind to prospectively assess the effects of an intensive lifestyle intervention on plasma and stool metabolites and the risk of T2D, using the state-of-the-art LC-MS platform and cutting-edge bioinformatics and statistical methods. Objective measures will be used to assess functional fitness (a 30-second chair sit- and-stand (CSS) test), physical activity (accelerometers), body composition (DXA), and extra-virgin olive oil consumption (urinary tyrosol and hydroxytyrosol levels). This application has the potential to advance our understanding of T2D pathophysiology and inform precision nutrition and T2D prevention. It has important implications for the US population because it may provide further evidence to strengthen current recommendations on the MedDiet as a part of a healthy lifestyle for chronic disease prevention.