A number of studies have shown that ingestion of crystalline amino acids, in particular branched chain amino acids (BCAA) can affect endurance performance, markers of muscle damage, and possibly mental focus. It seems that the longer and lower intensity events are affected more by consumption of BCAA. This study will test the hypothesis that BCAA availability limits central fatigue (the feeling of tiredness). While various aspects of performance have been tested no study to date has tested multiple exercise, muscular, and mental performance aspects in a single study. Thus, the overall purpose of this study is to determine whether an amino acid supplement changes the mental and physical response to prolonged exercise versus a placebo (carbohydrate drink).

This nutritional supplement could be useful on two levels. Improved mental focus and endurance performance and decreased central fatigue and muscle soreness could be valuable for enhancing athlete performance. Similarly, decreased central fatigue and muscle soreness may increase the willingness of the general public to exercise which could translate into decreased health risks and increased health benefits.

Specific Purpose and Hypotheses

In the current proposal we outline a study that will examine a comprehensive battery of tests to assess the efficacy of Amino Vital's Amino Acid (AA) formulation to improve physical and mental performance after exercise.

We hypothesize:

1. Ingestion of the Amino Acid (AA) formula will improve cognitive, motor, and physical performance on the post-exercise tasks in a hierarchical manner: 2AA > AA > placebo
2. Blood concentrations of amino acids will be higher in the AA trials in a hierarchical manner: 2AA >AA > placebo
3. Markers of muscle damage will be lower than placebo in a hierarchical manner: placebo > AA > 2AA

Recruitment Methods and Process Recruitment methods will be passive. Participants will be made aware of the study through the use of Hamilton Health Science/Faculty of Health Sciences Research Ethics Board (HHS/FHS REB) approved posted flyers at McMaster University and will voluntarily initiate contact with the study team. A recruitment poster will also be submitted for posting on the McMaster Daily News webpage.

Research Design and Methodology This study will use a double-blind repeated measures study design in which subjects will complete 7 sessions. The sessions consist of familiarization and the completion of three trials (placebo, AA, 2AA of AminoVital's formulation). Trials will be completed in a randomized, counterbalanced and blocked fashion such that with 12 subjects, 2 participants complete each trial order: ABC, ACB, BCA, BAC, CAB, CBA.

In the first session, participants will be screened using a standard health questionnaire. Interested participants will sign a consent form. They will then undergo testing of maximal oxygen consumption (VO2peak) using a treadmill. Subjects will also familiarize themselves with the physical and mental performance tests: Stroop test, profile of mood states (POMS), isometric strength test and an isokinetic muscle fatigue test. Following this initial session, participants will complete 3 trials (placebo, AA or 2AA drink formula) in random order.

On the first day of a trial, participants will arrive at the lab in the morning after a day of dietary control and a standardized breakfast including the treatment beverage. Prior to beginning exercise, participants will complete the physical and mental performance tests. A small catheter (flexible plastic tube) will be inserted into a forearm vein by Dr. Phillips or a medically trained and qualified member of the laboratory group; Dr. Tarnopolsky is responsible for the individuals inserting the venous catheters. The needle will be removed promptly after the insertion of each catheter; only the catheters will remain in their arms until the end of the trial. The catheter will be used for repeated blood sampling over the next six hours (see Figure 1). Additionally, breath samples will be taken at the same time as the blood samples. Following the performance tests, and prior to the start of exercise, a baseline blood sample will be drawn. Participants will then perform a bout of endurance exercise that consists of treadmill walking for 180 minutes at 40% of their peak VO2. Following the start of exercise, participants will consume a treatment beverage (or placebo) every 20 minutes after the start of exercise and continuing until 20 minutes after exercise (see Figure 1). Following the exercise bout, participants will repeat the physical and mental performance tests.

On the second day of a trial, participants will arrive after having consumed a standard breakfast including the treatment beverage. They will complete the physical and mental performance tests and one blood sample (4 mL) will be taken.

The trial day breakfast will consist of energy estimated for each subject based on the Harris-Benedict equation with an activity factor of 30%. The diet will be 55:30:15 in terms of percent energy from carbohydrate:fat:protein. Subjects will consume the same diet on the days prior to each exercise trial to control for pre-study calorie and carbohydrate/fat intake, which could affect substrate oxidation. Subjects will refrain from consumption of alcohol and caffeine on the day of the trial. Trials will take place at least 5d (but no more than 10d) apart and will commence at a time between ~6-10am and will proceed according to the schematic timeline shown in figure 1. The testing time for subjects will remain consistent throughout the study. We also believe that it is prudent for subjects to consume a fixed and constant volume of water (500 ml with breakfast) during all trials to prevent dehydration and to allow subjects to complete the exercise in safe manner.

Data Analyses/Statistics All data will be analyzed using a repeated measures two-way analysis of variance with time as a within factor and dose as a between factor. Significant ANOVA effects will be further scrutinized using Tukey's test as a post-hoc procedure. Where significant correlation of one variable with another exists, an analysis of covariance may also be performed. Significance will be accepted at P<0.05.

Instrumentation Sample analysis will be carried out using a microplate reader/ELISA for creatine kinase (CK), C-reactive protein (CRP), lactate dehydrogenase (LDH) and myoglobin, glucose and lactate auto-analyzers, High Performance Liquid Chromatography (HPLC) for amino acids as well as a metabolic cart gas analyzer to analyze breath samples.

Inclusion Criteria:

• Male
• Aged 18 to 30 years old
• Non-obese (body mass index ≤ 30 kg/m2)
• Non-smoker
• Healthy (see exclusion criteria below)

Exclusion Criteria:

• Any acute or chronic illness, cardiac, pulmonary, liver, or kidney abnormalities, uncontrolled hypertension, insulin- or non-insulin dependent diabetes or other metabolic disorders - all ascertained through medical history screening questionnaire.
• Arthritic conditions
• Individuals who cannot refrain from consuming any alcoholic beverages during the study
• Individuals who chronically consume any analgesic or anti-inflammatory drug(s), prescription or non-prescription
• A history of neuromuscular problems
• Individuals on any medications known to affect protein metabolism (i.e. corticosteroids, non-steroidal anti-inflammatories, or prescription-strength acne medications)