The purpose of this protocol is to investigate the role of expired non-metabolic carbon dioxide in the relationship between fatigability and recovery and the response to aerobic exercise training in healthy individuals. Both fatigability and recovery are profoundly influenced by mitochondrial energetics which can be inhibited by ionic by-product accumulation during exercise. Buffering mechanisms of these fatigue-inducing ions releases non-metabolic carbon dioxide (CO2) that can be measured as expired CO2 (VCO2) during cardiopulmonary exercise testing (CPET), however the role of non-metabolic VCO2 in the relationship between fatigability and recovery has yet to be investigated.

Furthermore, this study aims to identify the how the patterns of proteins in healthy individuals respond to aerobic exercise training (e.g. stationary cycling) over approximately one month. The underlying mechanisms of recovery after physical activity, including mechanisms or biological pathways that could be highlighted by analysis of proteins in urine, could add to scientific knowledge regarding physical activity tolerance and potential exercise interventions. This knowledge could eventually assist with designing precise and personalized exercise interventions to improve physical activity performance.

The investigators hypothesize that 1) non-metabolic CO2 will be at least moderately associated with the inverse relationship between fatigability and recovery; and 2) highly active adults, compared to sedentary individuals, will exhibit differential proteomic patterns in response to an initial acute bout and subsequent repeated bouts of aerobic exercise.

Subjects will be recruited from the greater Washington D.C. metro area by word of mouth, university classes, healthcare provider referral, social media posting, and by posted fliers. Healthy males and females as determined by the Physical Activity Readiness Questionnaire Plus (PARQ+) will qualify to participate, regardless of their fitness level. The study design and participation will be explained to those who are potentially interested in participating in the study. Individuals interested in participating as subjects will complete the PARQ+ and those answering "no" to all of the PARQ+ questions will qualify for inclusion. Those answering "yes" to one or more of the questions will be asked follow-up questions to determine if they meet inclusion/exclusion criteria. Subjects will then be consented and enrolled for participation.

Visit 1: Subjects meeting all inclusion criteria and no exclusion criterion will be consented and enrolled in the study. Subjects will then complete the International Physical Activity Questionnaire (IPAQ) to describe their current levels of physical activity. Height and weight measurements of the subject will also be taken. Subjects will then complete a standard peak cardiopulmonary exercise test (pkCPET) to volitional exhaustion with near infrared spectroscopy (NIRS) assessment of muscle oxygenation and microvascular reactivity, bioimpedance cardiographic (ZCG) assessment of cardiac output and stroke volume, and electrocardiographic (EKG) measurement of heart rate (HR) at rest and during exercise. After a 10-minute passive recovery period, subjects will perform an endurance based CPET (enCPET) at intensity of 70% of the peak wattage reached during the pkCPET, again to volitional exhaustion followed by a final 10-minute passive recovery period to conclude day one of testing.

Visit 2: Subjects will complete a submaximal square-wave test (swCPET) for measurement of oxygen on-kinetics. After a 10-minute recovery period, subjects will complete the same enCPET they performed during Visit 1 testing. This testing will again be followed by a 10-minute recovery period. EKG measurements of HR will be taken during exercise and rest periods. Subjects will receive a urine collection cup to be used prior to visit 3. Subjects will be asked to collect approximately 75-90 mL of urine on the morning of Visit 3 to provide upon arrival. Subjects will be asked to log food intake using the form described below for 48 hours, starting 24 hours prior to Visit 3.

Visits 3-19: On days 3-19, subjects will complete a continuous high intensity aerobic exercise training (AET) protocol. Subjects will warm up for approximately 5-minutes, exercise within their predetermined HR range for 45 minutes, followed by a 5-10 min recovery period. HR will be monitored using a Polar chest strap worn by the subject and a paired watch and the heart rate reading on the cycle ergometer monitored by the investigators. The entire training session will take approximately 60 minutes. Following Visit 3, subjects will be provided with a 2nd urine sample cup and asked to collect a "first-morning" urine sample (75-90mL) at home on the day after visit 3. Subjects will be asked to provide subsequent first-morning midstream urine samples at home on the morning of and the morning after visits 7, 11, 15, and 19 (10 total urine samples). Subjects will be provided with a copy of their initial food log and asked to repeat their nutritional intake for the same timeframe as the initial sample for each subsequent sample (24 hours prior to pre-exercise sample until post-exercise sample).

Visit 20: Subjects will repeat the same procedures performed at Visit 1 including a pkCPET, 10-minute recovery, enCPET, 10-minute recovery, in that order. NIRS, ZCG, and EKG again will be collected throughout both the active and recovery portions of the testing.

Visit 21: Subjects will repeat the same procedures performed on day two of testing including a swCPET, 10-minute recovery, enCPET, 10-minute recovery, in the order. EKG data will again be collected during the active and recovery portions of the testing.

Inclusion Criteria:

• age 18-60
• body mass index > 19 to <35 kg/m2
• able to pedal leg cycle ergometer
• able to comprehend and speak English

Exclusion Criteria:

• diabetes mellitus
• significant pulmonary dysfunction (eg. chronic obstructive lung disease; interstitial lung disease)
• hypertension
• anemia
• stroke
• cancer (other than melanoma)
• cardiac, pulmonary, thyroid, autoimmune, musculoskeletal, neurological, metabolic bone, mitochondrial, hepatic, renal, and/or psychiatric disease
• abnormal blood lipids
• active substance abuse or cognitive impairment
• chronic infection requiring antiviral or antibiotic treatment
• taking any medications that may limit exercise capacity or the ability to adapt to aerobic exercise training
• previously or currently on anticoagulant therapy or therapeutic hormone replacement/supplementation (excluding birth control)
• pregnant
• smoking