This randomized, parallel, controlled study, will investigate the effect of regular exercise on GM composition, inflammatory status, and insulin sensitivity, in the progression from normal glucose tolerance (NGT), to prediabetes (pre-D), to type 2 diabetes (T2D). Following baseline assessment of glucose tolerance, the participants will be randomly assigned to either a 12-week, thrice-weekly exercise training program followed by 4 weeks of detraining, or will remain sedentary for the 16-week intervention. Thus, the six study groups will be: 1) NGT group (NGT), NGT individuals - no exercise, 2) NGT exercise group (NGT+Ex), NGT individuals that will participate in training and detraining, 3) pre-D group (pre-D), pre-D individuals - no exercise, 4) pre-D exercise group (Pre-D+Ex), pre-D individuals that will participate in training and detraining, 5) T2D group (T2D), T2D individuals - no exercise, and 6) T2D exercise group (T2D+Ex), T2D individuals that will participate in training and detraining. Assessment of physiological measures, anthropometric characteristics, body composition, glucose tolerance, insulin sensitivity, complete blood count, lipidemic profile, GM composition, inflammatory status, oxidative stress, and muscle performance, will be conducted before and following 12 weeks of the exercise training intervention and following 4 weeks of detraining for all participants.

Type 2 diabetes is a global metabolic epidemic and a major global health threat. In 2021, 537 million adults aged 20-79 years worldwide had diabetes. T2D, is related with life threatening microvascular and macrovascular health complications that contribute tremendously to the burden of mortality and disability worldwide. T2D is defined by fasting hyperglycemia which is largely secondary to inadequate action of insulin. T2D is usually preceded by a state of intermediate hyperglycemia or Pre-D, which is characterized by impaired fasting glucose (IFG), impaired glucose tolerance (IGT), or both, and greatly increases the risk for T2D. In T2D, although insulin levels are normal or high, tissues such as liver, skeletal muscle, and adipose tissue become resistant to insulin resulting in high levels of blood glucose. T2D is associated with a systemic inflammatory response which seems to be an independent risk factor for the development of T2D. Additionally, individuals with T2D tend to have a more oxidative internal environment than healthy subjects. Hyperglycemia-induced oxidative stress has been found to affect the insulin signaling cascade and decrease GLUT4 gene transcription and also alter mitochondrial activity. T2D pathophysiology has also been associated with GM composition. Dysbiosis of GM is suggested to have a central role in the pathogenesis of insulin resistance and T2D through several mechanisms.

The important role of regular exercise for the prevention and treatment of T2D has been established. Most benefits of exercise on T2D management and prevention are realized through the adaptations to skeletal muscle which in turn induce acute and chronic improvements in insulin action. Exercise also exerts anti-inflammatory effects. Emerged evidence suggests that exercise may also favorably affect T2D by improving GM composition. The most promising effect of regular exercise is the alteration of GM towards a healthier microbial composition by producing a more diverse GM, decreasing pathogenic bacterial communities and increasing SCFAs-producing bacteria. However, the impact of exercise on the GM structure and function of T2D individuals is poorly understood, as only a limited number of studies exist in this area, so far.

According to a preliminary power analysis (a probability error of 0.05 and a statistical power of 80%), a sample size of 8-10 participants/group was considered appropriate to detect statistically meaningful changes between trials. Thus, ≥60 middle-aged individuals will be assessed for eligibility to participate in the study. The study will be conducted in a parallel, randomized, controlled design. The participants, will be primarily informed of the study procedures, as well as the benefits and possible risks, and they will also sign an informed consent form for participation in the study. All eligible individuals will provide blood samples for the determination of fasting blood glucose, glycosylated hemoglobin (HbA1c), and fasting plasma insulin, and will take an oral glucose tolerance test (OGTT) to determine their glycemic profile, according to which, they will be characterized as normal glucose tolerance (NGT) individuals, or pre-diabetes (pre-D) individuals, or type 2 diabetes (T2D) individuals. Physical activity levels will also be assessed through the International Physical Activity Questionnaire (IPAQ). Afterwards, the participants of each glucose tolerance stage will be randomly assigned to either the 12 weeks of regular combined aerobic and resistance exercise according to the guidelines for pre-D and T2D individuals, or remain sedentary for 12 weeks. Thus, six intervention groups will be as follows: i) NGT group (NGTG), NGT individuals - no exercise, ii) NGT exercise group (NGTG+Ex), NGT individuals that will participate in training and detraining, iii) pre-D group (pre-DG), pre-D individuals - no exercise, iv) pre-D exercise group (Pre-DG+Ex), pre-D individuals that will participate in training and detraining, v) T2D group (T2DG), T2D individuals - no exercise, and vi) T2D exercise group (T2DG+Ex), T2D individuals that will participate in training and detraining. Randomization of the conditions will be done by a software generating random integers available on the internet (Random.org).

Baseline measurements will take place at the Laboratory of Biochemistry, Physiology and Nutrition of Exercise (SmArT Lab), Department of Physical Education and Sports, University of Thessaly: physiological measures (resting heart rate, resting systolic and diastolic blood pressure, resting metabolic rate), anthropometric characteristics (body height, body mass, body mass index), body composition (amount of body fat, lean body mass, fat mass, bone density), muscle performance [aerobic capacity (VO2max), isokinetic strength of the lower extremities (isometric, concentric and eccentric torque of the knee extensors and knee flexors), handgrip strength, muscle power (countermovement jump)]. Additionally, the participants will provide feces for the determination of GM composition and GM metabolites [short chain fatty acids (SCFAs)], as well as blood samples for the determination of complete blood count (CBC), systemic inflammation [tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), C-reactive protein (CRP), zonulin, lipopolysaccharides-binding protein (LBP)], blood redox status [total antioxidant capacity (TAC), catalase (CAT), protein carbonyls (PC), reduced glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG ratio, malondialdehyde (MDA), uric acid, bilirubin], and lipid profile [total cholesterol (CHO-T), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TG)]. In addition, the participants will record their diet via a 7-days recall before their participation in the first experimental condition, and dietary data will be analyzed. All of the above measurements will be repeated following the 12-weeks of exercise intervention, as well as following the 4-weeks of detraining period. The participants that will be allocated into the regular exercise, except for VO2max, they will further undergo estimation of maximal strength of the main muscle groups for the determination of intensity of each participant's exercise program; aerobic capacity and muscle strength determination will also be repeated after four weeks of the regular exercise for the necessary intensity adjustments of the exercise program.

Inclusion Criteria:

• Age between 45 and 65 years old
• Sedentary lifestyle or insufficient daily physical activity according to guidelines for T2D
• Abstinence of anti-inflammatory drugs and/or antibiotics and/or dietary supplements that could affect GM composition before the study (>3 months)
• No other chronic diseases and/or musculoskeletal injuries (>6 months)

Exclusion Criteria:

• Age <45 years or >65 years
• Physically active individuals
• Consumption of anti-inflammatory drugs and/or antibiotics and/or dietary supplements that could affect GM composition before the study (<3 months)
• Other chronic diseases ot recent history of musculoskeletal injury (<6 months)