Experimental Design
This study used a double-blind-placebo-controlled pre/post test 2-treatment experimental design. On days 1 and 29, subjects reported to the exercise lab for anthropometric collection and to perform an incremental treadmill running protocol. During the 28 day study, subjects were randomly assigned to consume a supplement containing either βA (6.0 g·d-1) or Placebo (PL) Maltodextrin (6.0 g·d-1). Pre- and post-supplementation testing took place at the same time of day for each subject and on the same equipment. Subjects were asked to fast for 2 hrs prior to each test. Subjects were asked to abstain from taking any other dietary supplements and to maintain their regular diet and exercise patterns for the duration of the study. Subjects were also required to abstain from caffeine or vigorous exercise for 24 hrs before exercise testing.
Anthropometric data were recorded in light exercise clothing and bare feet using a wall mounted stadiometer and calibrated digital scale (Tanita Body Composition Analyzer TBF-300A, Tanita Corp, Arlington Heights, IL). Subjects were connected to an automated metabolic measurement system (Parvomedics TrueMax 2400, Consentius Technologies, Sandy, UT) via mouthpiece and headset and fitted with a telemetric heart rate monitor (Polar F6, Finland) in seated position for resting variables prior to testing.
Participants performed 3 minutes of walking on the treadmill at 6.4 km.hr
-1 (4.0 mph) to acclimate to the apparatus. The treadmill was then set at a fixed 9.6 km.hr
-1 (6.0 mph) for the duration of the test. Every 3 minutes, the treadmill incline was increased by 2% grade. After stage 5, any remaining stages ensued at 3% grade increase (stages: 0%, 2%, 4%, 6%, 8%, 11%, 14%, 17%). The test continued until the participant reached volitional exhaustion. Oxygen uptake was obtained every 30 seconds (s) throughout the test. VO2
max was recorded as the highest 30 s average recorded prior to volitional exhaustion. Criteria for VO2
max was attainment of at least two or more of the following: reaching a plateau in VO2 (< 2.1 ml.kg
-1.min
-1 increase) the final two stages of the test, achieving a respiratory exchange ratio (RER ≥ 1.10) and/or reaching a HR within 5 beats per min
-1 of predicted maximal value (220 - age). In the final 30 s of each stage, participants were asked to report an overall body rating of perceived exertion (RPE) using a 6-20 numeric scale [
21], heart rate was recorded, and a capillary blood lactate sample was collected. Subjects were oriented to the RPE scale prior to initiation of the test.
A fixed marker of 4.0 mmol·L
-1 blood was used to define the onset of blood lactate accumulation (OBLA). This fixed lactate measurement provides the most reasonable and accurate lactate analysis relative to the scope of this study and has been shown to be a valid evaluation of physiological changes with specificity to endurance performance [
17], and improvements in endurance fitness [
18].
Immediately following RPE and HR data collection with 30 s remaining in each stage, subjects while running were asked to extend their left index finger, which was prepared with an alcohol pad and dried with gauze. After preparation, a lancet device was applied to the fingertip and samples were collected in capillary tubes. All lactate samples were immediately analyzed in duplicate using an Accutrend Lactate Analyzer (F. Hoffman-La Roche Ltd, Basel, Switzerland). After compiling the data, the stage that elicited 4.0 mmol/L blood lactate which has been previously identified as the OBLA [
22] was used to determine lactate threshold. OBLA,
VO2max@OBLA and HR@OBLA were all calculated using linear interpolation between relevant data points as has been previously explained by Neville et al. [
23].
The treadmill protocol continued until volitional exhaustion was attained and the highest heart rate experienced during the test was recorded as Max Heart Rate (MHR). OBLA was then also identified by the percentage of maximum heart rate (%MaxHR@OBLA) at which it occurred.
Supplementation
During the study, subjects were asked to refrain from taking any other dietary supplements or making changes to their regular dietary and exercise patterns. The participants were randomly assigned in a double-blind manner to receive either β-Alanine or Placebo. The supplements were provided to the participants in identical, unmarked, sealed containers, supplied by Athletic Edge Nutrition, Miami, Florida. Subjects received βA supplement (6.0 g·d-1 βA, 600 mg N-Acetylcysteine, 2.7 mg alpha-lipoic acid, 45 IU Vitamin E) or a PL (6.0 g·d-1 Rice Flour Maltodextrin). Both groups followed the same supplementation protocol of 3 capsules 3 times daily with meals.
Supplementing with 6.4 g·d
-1 of βA for 28 days has been shown to increase carnosine levels by 60% [
4,
12] so it can be assumed that supplemented subjects in this study experienced a significant increase in intramuscular carnosine concentration. Three of the eight subjects in the βA supplemented group reported tingling in their fingers and hands. No other side effects were reported by those individuals supplemented with βA and subjects in the PL group reported no side effects.
Statistical Analysis
Because of the degree of non-normality in the distributions, data transformation could not be done to obtain statistical normality. For this reason, nonparametric statistical methods were used to analyze the data. The Friedman test was used to determine within group differences; and the Mann-Whitney test was used to determine between group differences. Data were analyzed using SPSS for Windows (Version 16.0, 2007 Chicago, IL) Prior to initiation of the study the alpha level was set at p < 0.05 to determine statistical significance. Data are presented as means ± standard error (SE).