Twenty-two aerobically trained and nineteen untrained male and female subjects (26.1 ± 7.6 yrs, 172 ± 8.7 cm, 73.5 ± 17 kg, and 21.2 ± 7.0%) participated in this study. Aerobically trained individuals had been training a minimum of two years for an average of 8 ± 2 hours/week, 9 ± 3 workouts/week, and averaged 68 ± 36 kilometers/week of aerobic exercise (i.e., running, cycling and/or swimming). Untrained individuals did not participate in any regular form of aerobic exercise for at least the past year.
All subjects signed informed consent documents and the study was approved by the Baylor University Institutional Review Board prior to any data collection. Subjects were not allowed to participate in this study if they reported any of the following: 1) current or past history of anabolic steroid use; 2) any metabolic disorders or taking any thyroid, hyperlipidmeic, hypoglycemic, anti-hypertensive, or androgenic medications; 3) ingested any ergogenic levels of creatine, HMB, thermogenics, ribose, pro-hormones (i.e., DHEA, androstendione, etc.) or other purported anabolic or ergogenic nutritional supplements within 2 months prior to beginning the study and to not take any additional nutritional supplement or contraindicated prescription medication during the protocol.
Figure 1 shows the experimental design used in this study. The study was conducted in a randomized, double-blinded, and placebo-controlled manner. All subjects eligible to participate in the study completed a familiarization session where they were provided information (both verbal and written) regarding the study design, testing and supplementation protocols. Informed consent, medical documents and training history questionnaires were also completed at this time. Subjects were then required to perform an isokinetic leg extension test and 30-second anaerobic Wingate test to become familiar with the protocol and equipment. The procedures for all exercise tests are described in detail below. Following the practice trials, subjects were scheduled to return 48 hours later for baseline testing. Subjects were asked to not change their dietary habits in any way throughout the study. This was monitored by having each subject document dietary intake for 4 days (3 weekdays and 1 weekend day) before each testing session. In addition, each subject was instructed to fast for 12 hours and not to perform any physical activity for the 48 hours preceding each testing session.
At baseline testing (T1), subjects reported to the lab in the morning to complete questionnaires regarding health status. Subjects then performed three exercise tests in the following order: 1.) isokinetic knee extension muscle endurance test; 2.) 30-second Wingate anaerobic capacity test; and, 3.) a maximal cardiopulmonary graded exercise test. Thirty minute rest periods were allocated between each exercise test. Following baseline measurements, subjects were matched based on age and aerobic capacity and randomly separated into two groups: 1.) a dextrose placebo group; or 2.) a fast-melt CoQ10 group [Fast-Melt Coenzyme Q10, Pharma Base, S.A. (Switzerland)]. Subjects were instructed to return to the lab in 48 hours for testing and ingestion of the first supplement.
On returning to the lab (Day 0, T2), subjects completed the questionnaires. Prior to ingestion of the first supplement dose, body mass, total body water, and body composition measurements were obtained. Subjects then donated approximately 20 ml of fasting blood and approximately 15–30 mg of muscle using standard procedures described below. Subjects consumed either the placebo or CoQ10 supplement. At 60 minutes following ingestion of the supplement, subjects performed the three exercise tests as mentioned above with 30 minutes of recovery between each exercise test. Additional blood samples were taken immediately following each exercise test. Subjects returned to the lab following 2 weeks of supplementation to complete the same battery of assessments. No supplement was ingested on day 14 (T3) prior to performing the exercise tests.
Muscle endurance assessment
At baseline, day 0 and 14, subjects performed a 50-repetition Isokinetic leg extension test. Prior to testing, subjects warmed up on a stationary bicycle for 2–3 min at a 50–60 rpm. Subjects then performed five warm-up repetitions on the Biodex (Biodex Medical Systems, Shirley, NY, USA) isokinetic leg extension machine. After these warm-up repetitions, subjects performed unilateral leg extensions (right leg) for one set of 50 repetitions  at 180 degrees/s. Test-test reliability of this test has been shown to be 0.78 to 0.82 .
Anaerobic capacity assessment
At baseline, day 0, and day 14, subjects performed a 30-second Wingate anaerobic capacity test 30 minutes following completion of the muscle endurance test. Subjects warmed up for 2 min at 50–60 rpm on a stationary bicycle ergometer before performing the Wingate test. This warm-up was continued into the start of the sprinting portion of the Wingate test, which allows for a flying start. The Wingate anaerobic capacity test was performed on the LODE cycle ergometer (Amsterdam, Netherlands) with a resistance of 0.7 Nm/kg. Test-to-test variability in performing repeated Wingate tests in our laboratory yielded correlation coefficients of r = 0.98 ± 15% for mean power.
At baseline, day 0, and 14, subjects performed a volitional maximal cardiopulmonary exercise test according to the Bruce protocol 30 minutes following the anaerobic capacity test . Participants were instructed to perform the test for as long as possible to ensure a true maximal attempt. Standard ACSM test termination criteria were monitored and followed throughout each test . Metabolic gases were obtained with the Parvo Medics 2400 TrueMax metabolic measurement system (Sandy, UT, USA) on a Trackmaster TMX425C treadmill (Newton, KS, USA). The mean coefficient of variation (assessing maximum oxygen consumption) for this protocol was 6.5% (range, 2–14%) . Ventilatory threshold (VANT) was expressed as a percentage maximal oxygen consumption (mL·kg-1·min-1). To determine VANT, data from the graded exercise test for minute ventilation (VE), ventilation equivalent of VO2 (VE/VO2), and ventilation equivalent of VCO2 (VE/VO2) were plotted against exercise time (i.e. workload) and analyzed manually by an experienced exercise physiologist in a blinded manner.
Subjects were assigned in a double-blind and randomized manner to ingest a dextrose placebo (CON, n = 20) or fast-melt CoQ10 formulation (CoQ10, n = 21). Subjects were matched into one of two groups according to age and aerobic capacity. Subjects were required to initially ingest 2 capsules (100 mg) prior to exercise performance testing on day 0. Following testing on day 0, subjects continued consuming 100 mg twice daily (morning and night) for 14 days. Supplements were prepared in tablet form and packaged in generic bottles for double blind administration by Pharma Base, S.A. (Switzerland). Supplementation compliance was monitored by having the subjects return empty bottles of the supplement at the end of 14 days of supplementation.
Prior to each testing session, subjects were instructed to record all food and fluid intake over a 4-day period, which was reflective of their normal dietary intake and to include one weekend day. Dietary inventories were then reviewed by a registered dietician and analyzed for average energy and macronutrient intake using the ESHA Food Processor (Version 8.6) Nutritional Analysis software (Salem, OR, USA).
At the beginning of every testing session, subjects had their height and body mass measured according to standard procedures using a Healthometer (Bridgeview, IL, USA) self-calibrating digital scale with an accuracy of ± 0.02 kg.
Total body water was estimated using a Xitron 4200 Bioelectrical Impedance Analyzer (San Diego, CA). Whole-body composition measurements (excluding cranium) were determined with a Hologic Discovery W Dual-Energy X-ray Absorptiometer (DEXA; Hologic, Bedford, MA, USA) by using procedures previously described [26, 27]. Test-retest reliability studies performed on male athletes with this DEXA machine revealed mean deviation for total bone mineral content and total fat free/soft tissue mass of 0.31% with a mean intraclass correlation of 0.985.
Subjects were required to fast for 12 hrs prior to donating approximately four teaspoons (20 milliliters) of venous blood from an antecubital vein using standard phlebotomy procedures. Blood analyzed for CoQ10 was placed in a test tube containing heparin as the anticoagulant and immediately centrifuged at 3000 g using a standard bench top centrifuge (Cole Palmer, Vernon Hills, IL, Model # 17250-10) for 15 minutes at room temperature. Plasma was separated and stored at -80°C in polypropylene tubes for later analysis. Blood analyzed for makers of oxidative stress and clinical chemistry panels were placed into two serum separation tubes and immediately centrifuged at 1100 g for 15 min. Serum was separated and stored at -80°C in polypropylene tubes for later analysis. Finally, blood was collected in a single lavender top tube containing K2 EDTA and refrigerated for approx 4–6 h for subsequent complete blood count with platelet differentials analysis (hemoglobin, hematocrit, red blood cell counts, MCV, MCH, MCHC, RDW, white blood cell counts, neutrophils, lymphocytes, monocytes, eosinophils, baosphils) using an Abbott Cell Dyn 3500 (Abbott Laboratories, Abbott Park, IL) automated hematology analyzer. Whole blood was only analyzed pre- and post-exercise.
Clinical chemistry analysis
Serum samples were analyzed using a Dade Behring Dimension RXL (Deerfield, IL, USA) automated clinical chemistry analyzer that was calibrated and optimized according to manufacturer guidelines. This analyzer has been known to be highly valid and reliable in previously published reports . Each serum sample was assayed for a standard complete metabolic panel [(glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin, globulin, sodium, chloride, calcium, carbon dioxide, total bilirubin)], lipid panel [(triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol:HDL)] and clinical markers of protein and fatty acid metabolism [(uric acid, creatinine, blood urea nitrogen (BUN), BUN:creatinine ratio, total protein, creatine kinase (CK), ketones (betahydroxybutyrate), and lactate dehydrogenase (LDH)]. Test to test reliability (within and between) of performing these assays ranged from 2 to 6% for individual assays with an average C
of ± 3%. Samples were run in duplicate to verify results if the observed values were outside control values and/or clinical norms according to standard procedures.
Markers of oxidative stress
Serum samples were assayed using standard commercially available spectrophotometric assay kits (Caymen Chemical Company, Michigan, USA) for Thiobarbituric Acid Reactive Substance (TBARS, Malondialdehyde-MDA) and Superoxide Dismutase (SOD). Enzyme immunoassay (EIA) was used to measure serum 8-Isoprostane. Serum concentrations were determined using a Wallac-Victor IV (Perkin-Elmer Life Sciences, Boston, MA) micro plate reader at an optical density of 450 nm against a known standard curve using standard procedures. Test to test reliability (within and between) of performing these assays ranged from 3 to 6% for individual assays with an average C
of ± 4%.
Muscle sample collection
Fine Needle Aspiration (FNA) muscle biopsies were taken on days 0 and 14 prior to, and following performance tests in order to examine total CoQ10 content in the muscle according to standard procedures . Participants were instructed to refrain from exercise 48 h prior to each muscle biopsy. Muscle was extracted from the lateral portion of the vastus lateralis midway between the patella and iliac crest of the dominant leg using a TRU-CORE® 1 Automatic Reusable Biopsy Instrument (Angiotech, Medical Device Technologies, INC., Gainsville, Florida, USA). Briefly, 0.5 ml of 1.0% Lidocaine HCl was injected subcutaneously prior to making a small pilot hole with a 23 gauge sterile needle. The biopsy needle was placed into the biopsy device and the inserted into the previously made pilot hole (a depth of about 5–10 mm). A muscle sample was obtained by the activation of a trigger button, which unloaded the spring and activated the needle to collect a muscle piece. The biopsy needle was removed from the pilot hole and the muscle specimen was removed from the biopsy needle using a sterile scalpel and immediately frozen in liquid nitrogen and stored at -80°C for further analyses. This whole biopsy procedure was repeated two to three times in order to obtain sufficient muscle tissue (15–30 mg).
Frozen plasma and muscle samples were sent to Craft Technologies (Wilson, NC, USA) for assay of plasma and muscle CoQ10 concentrations using methods described by Tang et al.,  and Rousseau and Varin . Total CoQ10 was determined by summing the oxidized and reduced forms.
Data are presented in all tables and throughout the text as mean (± SD). Plasma and muscle CoQ10 levels and markers of oxidative stress were analyzed using 2 × 2 × 2 × 4 (group × training status × gender × exercise [Baseline, Isokinetic, Wingate, Cardiopulmonary]) repeated measures ANOVA to assess the changes in plasma and muscle CoQ10 levels and markers of oxidative stress following acute (T2) and chronic (T3) supplementation. Delta scores (post minus pre values) were calculated for each exercise test (Isokinetic, Wingate, Cardiopulmonary) and analyzed using repeated measures ANOVA. All remaining data were analyzed using 2 × 2 × 2 × 3 (group × training status × gender × day [baseline, day 0 and 14]) repeated measures ANOVA. LSD pairwise comparisons were used to analyze any significant group × time interaction effects. Pearson product correlations were used to determine any relationship between criterion variables and an alpha level of 0.05 was adopted throughout to prevent any Type I statistical errors.