Exercise and Protein Efficiency in T2D

1. Enrollment, Informed Consent, and Screening Questionnaires: Prior to study enrollment, participants will be asked to read a digital copy of an informed consent document provided using the e-Consent framework through the Research Electronic Data Capture (REDCap) system. Should participants have any questions about the study after reading the document, they will be encouraged to contact the research team prior to providing informed consent. Upon providing informed consent, participants will receive an email containing questionnaires designed to assess study eligibility via REDCap. Specifically, participants will be asked to complete a health and medical history questionnaire, physical activity readiness questionnaire (PARQ), International Physical Activity Questionnaire Long Form (IPAQ), and Godin-Shephard Leisure-Time Physical Activity Questionnaire (LTPAQ). Information from the questionnaires will be used to assess exclusion criteria, and the IPAQ will be used to assess baseline habitual activity level. Upon completion of these questionnaires, the research team will review the responses and determine eligibility.
2. Preliminary Testing Day 1:

2.1 Preparation: Participants will arrive for the preliminary testing session after an overnight fast (~10 h no caloric foods/beverages consumed) and having refrained from exercise for the 72 h prior.

3.2 Cardiorespiratory fitness assessment: Peak aerobic capacity and maximum heart rate (HR) will be estimated using a submaximal graded stationary bicycle exercise test. Briefly, participants will pedal for 3 minutes at a resistance of 0kg, maintaining a cadence of 50 as a warm-up. Then, the investigators will have the participant pedal for 3-4 minutes, or until a steady state is reached, at 0.5kg, maintaining the same cadence. Resistance is increased every 3-4 minutes until the participant reaches 85% of age-predicted maximal heart rate (220-age).

3.3 Muscle Performance (1RM and power Testing): maximum strength will be assessed using a 10-repetition max (10-RM) on a leg extension, leg press, leg curl, chest press, shoulder press, seated pulldown, and seated row machines. Following a 5-minute warm-up on a stationary bike at a self-selected resistance and speed, participants will perform a set of 8-10 reps at around 50% of what they think they can lift 10 times. 1-2 minutes of rest will be given between each try. The next set will be around 90% of what the participant believes they can lift ten times. investigators will gradually increase the weight by 2.5-5.0% at a time until the participant can no longer lift it. A two-minute rest will be provided between the maximum number of attempts and between exercises.

3.3.5 The HUMAC NORM Isokinetic Dynamometer (CSMi, Stoughton, Massachusetts) will be used to assess peak torque, average workload, and average power in concentric tests and maximal voluntary contraction (MVC). Each participant will be seated upright, with the knee bent. The shank of the dominant leg will be secured to a padded, rigid metal bar using a Velcro strap. With standardized verbal encouragement, participants will perform brief maximal voluntary isometric contractions (MVC), separated by at least 60 seconds of rest, to determine maximal knee extension torque. Maximal power of the knee extensor muscles will be obtained at several test velocities using 2 sets of 4 maximal isokinetic knee extensions, including at a velocity of 60 deg/s. Strong verbal encouragement will be provided to kick as hard and as fast as possible throughout the full range of motion for each velocity. Participants will rest for at least 60 seconds after each set of 4 contractions to minimize muscle fatigue during the power assessment.

1. Preliminary testing day 3: Dietary Efficiency trial 4.1 Preparations: 3 days prior to the trial, participants will receive 3 days' worth of standardized meals and be instructed to prepare meals based on the manufacturer's instructions. Participants will arrive for the preliminary testing session after an overnight fast (~10 hours, no caloric foods/beverages consumed) and after refraining from exercise for the 72 hours prior.

5.2 Blood Collection: An intravenous catheter will be placed in a hand or arm vein for repeat blood sampling. Blood samples will be collected at 10 time points (-30, 0, 30, 60, 90, 120, 150, 180, 240, and 300 minutes) during each gut microbiome trial (pre- and post-training).

5.3 Mediterranean-based modeled meal: At time point 0, participants will be asked to ingest ~20g of protein enriched with L-[ring- 13C6] Phenylalanine to ~30 MPE. The amino acid and fat composition of this meal will be modeled based on ~3.05 oz of salmon filet.

1. Fecal Collection A fecal sample collection kit will be provided to participants, and they will be asked to collect the first stool produced after the postprandial gut microbiota tracer trial (5.0). Participants will be asked to adhere to a three-day controlled diet prior to each of 2 fecal collections (1x pre-testing, 1x post-testing), and samples will be delivered to the lab at their next study visit.

7.0 Exercise Training: 7.1 Strength Training: On strength training days, participants will warm up on a stationary bike for 5 minutes before each training session. The training session will consist of 1-3 sets on the leg press, leg extension, and leg curl machines. In addition, participants will be asked to perform upper-body exercises on guided machines (seated chest press, seated shoulder press, seated row) on alternating training days. All sessions will be separated by 2-3 days. Training intensity will be progressive and based on individual comfort levels. During the first week of training (week 0), the training intensity will be gradually increased from ~60% 1 Repetition Max (1RM) (10-15 repetitions) to ~70% 1RM (10-12 repetitions).

7.2 Endurance training: For aerobic-based training, supervised exercise sessions will occur on a stationary bike. Exercise intensity will be controlled by heart rate (HR) and Rate of Perceived Exertion (RPE) using Borg's original scale (6-20 points). Exercise intensity will be progressive: weeks 1-3 will be 50-60% HR for 30 minutes; weeks 4-6, 50-60% for 40 minutes; weeks 7-9, 60-70% for 50 minutes; weeks 10-12, 60-70% for 60 minutes.

1. Psychological Evaluation During each in-person exercise day, participants will be evaluated using several psychological well-being questionnaires. The International Personality Item Pool (IPIP-50) is a 50-item assessment of the Five-Factor Model (Openness to Experience, Conscientiousness, Extraversion, Agreeableness, and Neuroticism/Emotional Stability). Additionally, the PRETIE-Q is a 16-item questionnaire designed to assess personal preferences for exercise intensity and perceived tolerance for it. The Dispositional Resilience Scale (DRS-15) will be used to assess participants' resilience, and the Hospital Anxiety and Depression Scale (HADS), a 14-item instrument used to assess the severity of anxiety and depressive symptoms in the past 7 days. The option to do these electronically or on paper will be given.