Subjects
Twenty-seven male (n = 18) and female (n = 9) endurance-trained runners or triathletes (21.8 ± 3.9 years, 67.4 ± 11.8 kg, 15.0 ± 6.0 % body fat, 51.2 ± 11.4 kg free fat mass) participated as subjects in this study. Subjects were recruited through paper and electronically distributed flyers at Texas A&M University. Entrance criteria required the runners or triathletes to have been involved in a consistent running program for at-least 1-year and able to run a half-marathon (21.1 km) in less than 2 h. Figure 1 provides a breakdown of the subject population. Subject discontinuation of participation was not related to any aspect of the supplementation or testing protocol.
All subjects signed informed consent documents and the study was approved by the Texas A&M 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) metabolic disorders or taking any thyroid, hyperlipidemic, hypoglycemic, anti-hypertensive, anti-inflammatory (e.g. NSAIDs), and/or androgenic medications; 2) history of hypertension, hepatorenal, musculoskeletal, autoimmune, and/or neurological disease(s); and 3) allergy to cherries or any cherry components (e.g. polyphenols, anthocyanins, anthocyanidins).
Experimental design
The study was conducted in a randomized, double-blind, and placebo-controlled manner (see Fig. 2). All subjects completed a morning familiarization (FAM) session where they were provided detailed information regarding the study design, testing procedures, and supplementation protocols. Informed consent, medical history, and endurance training history questionnaires were also completed during the FAM session. A nurse reviewed medical history documents and performed a physical exam (resting vital signs and lung auscultation) on each subject to ensure participation eligibility. A fasting blood sample was taken at the end of the FAM session. Approximately 10-d prior to the endurance exercise intervention, subjects returned to the lab for a morning baseline testing session to determine body mass, height, and body composition. Following baseline measurements subjects were matched based on average reported race pace, fat free mass, body mass, and age and randomly separated into two groups: 1) a placebo group or 2) a powdered tart cherry group. Subjects were instructed to not change their dietary habits in any way throughout the study. Nutritional habits were monitored through self-dietary recall for 4-d (3 weekdays and 1 weekend day) of the first seven supplementation days.
Subjects were instructed to begin supplementation 7-d prior to the endurance exercise challenge (Day 0). Subjects were asked to fast overnight for 10-h to account for diurnal variation as well as abstain from exercise and consumption of non-steroidal anti-inflammatory medications (NSAIDs) for 48-h prior to all testing days. On the day of the endurance exercise challenge, the subjects reported to the lab where body mass, resting heart rate, and resting blood pressure were measured. Subjects then donated a fasting venous blood sample (approximately 20 ml) using standard clinical procedures and rated perceptions of muscle soreness to a standardized application of pressure on their dominant thigh at three designed locations using a graphic pain rating scale (GPRS). Twenty minutes prior to the start of the half-marathon race, subjects were allowed to warm-up as they normally would before running a road race. Subjects completed a half-marathon (21.1 km) run outdoors at their normal race/competition pace. Both water and glucose-electrolyte drinks were provided ad libitum to the subjects at regular intervals during the race. Fasting (except 60-min post-run) blood samples and GPRS ratings of quadriceps muscle soreness were completed at 60-min, 24 and 48-h of post-run recovery. The last or tenth day of supplementation correlated with 48-hours post-run recovery.
Exercise protocol
Half-marathon (21.1 km) run
On the morning of supplementation day 8, all subjects performed an outdoor half-marathon run (21.1 km) for best time on a closed course under simulated race day conditions. Race start (0800) conditions were: ambient temperature = 22.8 °C, wind = 14.5 kph, humidity = 90 %, dew point = 21.1 °C. Conditions at the race finish (1030) were: ambient temperature = 25.0 °C, wind = 14.5 kph, humidity = 86 %. The race was run completely on concrete and pavement surfaces. All subjects were given 20-minutes for individual warm-up routines. At regular intervals (4 total locations) throughout the race, fluids (water and/or glucose-electrolyte beverages) were made available ad libitum to the subjects. Each subject had their own water and glucose-electrolyte beverage bottle labeled with a number that corresponded to their race number. All fluid bottles were weighed before and after the race to determine fluid consumption for each subject. Official race splits and finish times were recorded by designated lab staff. Following the race, subjects were not allowed to run to cool down, only stretching and minimal ambulation was permitted until the 60-min post-run testing session.
Supplementation protocol
Subjects were assigned in a double-blinded and randomized manner to ingest a rice flour placebo (P, n = 16) or powdered tart cherry (TC, n = 11). Subjects were matched into one of the two groups according to average reported race pace from previous (within the last 1 year) race events, fat free mass, body mass, and age. Subjects were instructed to ingest one 480 mg supplement capsule one time daily directly after breakfast at 0800 for 7-d prior to, the day of, and for 2-days following the half-marathon race for a total supplementation timeline of 10-d. The tart cherry supplements contained 480 mg of freeze dried Montmorency tart cherry skin powder derived from tart cherry skins obtained after juicing (CherryPURE™ Freeze Dried Tart Cherry Powder, Shoreline Fruit, LLC, Transverse City, MI, USA). Prior analytical testing conducted in 2012 by Advanced Laboratories (Salt Lake City, UT, USA) demonstrated that 31 mL (10.5 fl oz) of tart cherry juice provides approximately 600 mg of phenolic compounds and 40 mg of anthocyanins, which is equivalent to consuming 290 mg of CherryPURE™. Using the same comparison, the 480 mg CherryPURE™ supplement provided in the current study would be equivalent to 51.3 mL (17.4 fl oz) of tart cherry juice providing 991 mg of phenolic compounds and 66 mg of anthocyanins. The supplements were prepared for distribution by Shoreline Fruit, LLC and sent to Advanced Laboratories (Salt Lake City, UT, USA) to quantify the nutritional contents of the powdered tart cherry supplements. Both supplements were prepared in capsules identical in taste and appearance. The supplements were packaged in generic bottles by Shoreline Fruit, LLC for double blind administration.
Procedures
Dietary inventories
Within the first 7-d of supplementation, subjects were instructed to record all food and fluid intake over a 4-d period (3 weekdays, 1 weekend day). Dietary inventories were then reviewed by a registered dietician and analyzed for average daily energy (total kilocalories), macronutrient (protein, fat, and carbohydrates), and dietary antioxidant (vitamins C and E, and β-carotene) intake using ESHA Food Processor (Version 8.6) Nutritional Analysis software (ESHA Research Inc., Salem, OR, USA).
Anthropometrics and Body composition
At the beginning of every testing session, subjects had their height and body mass measured according to standard procedures using a Healthometer Professional 500KL (Pelstar LLC, Alsip, IL, USA) self-calibrating digital scale with an accuracy of ±0.02 kg. Whole body bone density and body composition measures (excluding cranium) were determined with a Hologic Discovery W Dual-Energy X-ray Absorptiometer (DEXA; Hologic Inc., Waltham, MA, USA) equipped with APEX Software (APEX Corporation Software, Pittsburg, PA, USA) by using procedures previously described [29]. Mean test-retest reliability studies performed on male athletes in our lab with this DEXA machine have revealed mean coefficients of variation for total bone mineral content and total fat free/soft tissue mass of 0.31–0.45 % with a mean intraclass correlation of 0.985 [30]. On the day of each test, the equipment was calibrated following the manufacturer’s guidelines for quality assurance.
Muscle soreness perception assessment
Pressure application to the three specified areas of the quadriceps muscle group on each subject’s dominant leg was standardized to 50 N of pressure using a handheld Commander Algometer (JTECH Medical, Salt Lake City, UT, USA). The standard amount of pressure was applied to the vastus lateralis at both 25 and 50 % of the distance between the superior border of the patella to the greater trochanter of the femur at the hip and to the vastus medalis at 25 % of the distance between the aforementioned landmarks. These three specific locations were measured and marked with a permanent marker on each subject during the baseline muscle soreness perception measurement before the half-marathon race. The subjects were asked to maintain these three marked locations between testing sessions to avoid error with secondary measurement. The subject was seated in a reclined supine position and given the algometer GPRS sheet to evaluate the perception of muscle soreness at each of the three quadriceps locations. The order of pressure application was standardized across all sessions and subjects: 25 % VM, 25 % VL, and 50 % VL. The 50 N of pressure was applied to a relaxed quadriceps at each of the three locations using the algometer for a period of 3-sec to give the subject enough time to record their soreness evaluation on the GPRS. Perceptions of muscle soreness were recorded by measuring the distance (centimeters) of the participant mark on the GPRS from 0 cm (no pain). Reliability statistical analyses revealed a mean intraclass correlation of 0.909.
Blood collection
Subjects donated approximately four teaspoons (20 mL) of venous blood after a 10-h fast from an antecubital vein using standard phlebotomy procedures. Blood samples were collected in two 7.5 mL BD Vacutainer® serum separation tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), left at room temperature for 15-min, and then centrifuged at 3500 rpm for 10-min using a standard, refrigerated (4 °C) bench top Thermo Scientific Heraeus MegaFuge 40R Centrifuge (Thermo Electron North America LLC, West Palm Beach, FL, USA). Serum supernatant was removed and stored at −80 °C in polypropylene microcentrifuge tubes for later analysis. The multiple serum microcentrifuge tubes for each subject was allocated for a specific group of assays and thawed only once during analysis. Blood was also collected in a single 3.5 mL BD Vacutainer® containing K2 EDTA (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), left at room temperature for 15-min, and refrigerated for approximately 3–4 h before complete blood count analysis.
Clinical chemistry analysis
Whole blood samples were analyzed for complete blood count with platelet differentials (hemoglobin, hematocrit, red blood cell counts (RBC), white blood cell counts (WBC), lymphocytes, granulocytes (GRAN), and mid-range absolute count (MID) using a Abbott Cell Dyn 1800 (Abbott Laboratories, Abbott Park, IL, USA) automated hematology analyzer. The internal quality control for Abbott Cell Dyn 1800 was performed using three levels of manufacturer control fluids to calibrate acceptable standard deviation (SD) and coefficients of variation (CV) values for all aforementioned analytes. Samples were re-run if the observed values were outside control values and/or clinical norms according to standard procedures. Reliability statistical analyses revealed a mean intraclass correlation of 0.729 across all measures. Serum samples were analyzed using a Cobas c111 (Roche Diagnostics GmbH, Indianapolis, IN, USA) automated clinical chemistry analyzer that was calibrated according to manufacturer guidelines. This analyzer has been known to be highly valid and reliable in previously published reports [31]. Each serum sample was assayed for a standard partial metabolic panel [(aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin)] and clinical markers of protein and fatty acid metabolism [(uric acid, creatinine, blood urea nitrogen (BUN), BUN:creatinine ratio, total protein, and creatine kinase (CK)]. The internal quality control for the Cobas c111 was performed using two levels of manufacturer control fluids to calibrate acceptable SD and CV values for all aforementioned assays. Samples were re-run if the observed values were outside control values and/or clinical norms according to standard procedures. Reliability statistical analyses revealed a mean intraclass correlation of 0.793 across all measures.
Markers of anabolic/catabolic hormone status
Serum samples were assayed using standard commercially available enzyme-linked immunosorbent assay kits (ELISAs) for cortisol and testosterone (ALPCO Diagnostics, Salem, NH, USA). Serum concentrations were determined calorimetrically using a BioTek ELX-808 Ultramicroplate reader (BioTek Instruments Inc., Winooski, VT, USA) at an optical density of 450 nm against a known standard curve using manufacturer recommended procedures. Samples were run in duplicate according to standard procedures. Test to test variability of performing these assays yielded average CV values for the aforementioned markers of: CORT (±6.85 %), and TEST (±4.47 %) with a test retest correlation for the same markers of: CORT (r = 0.92), TEST (r = 0.98).
Markers of oxidative stress
Serum samples were assayed using standard commercially available ELISA kits for Superoxide Dismutase (SOD Activity Assay kit), Total Antioxidant Status (TAS, Antioxidant Assay kit), Thiobarbituric Acid Reactive Substance (TBARS, Malondialdehyde-MDA, TCA method kit) (Cayman Chemical Company, Ann Arbor, MI, USA), and Nitrotyrosine (ALPCO Diagnostics, Salem, NH, USA). Serum concentrations for SOD and Nitrotyrosine were determined calorimetrically using a BioTek ELX-808 Ultramicroplate reader (BioTek Instruments Inc., Winooski, VT, USA) at an optical density of 450 nm against a known standard curve using standard procedures, while TAS serum concentrations were analyzed calorimetrically at 405 nm. Lastly, serum concentrations for TBARS were determined fluorometrically using a SpectraMax Gemini multimode plate reader (Molecular Devices LLC, Sunnyvale, CA, USA) at an excitation wavelength of 530 nm and an emission wavelength of 550 nm against a known standard curve using standard procedures. Samples were run in duplicate according to standard procedures. Test to test variability of performing these assays yielded average CV values for the aforementioned markers of: SOD (±8.35 %), TAS (±14.24 %), TBARS (±8.30 %), and NT (±10.03 %) with a test retest correlation for the same markers of: SOD (r = 0.83), TAS (r = 0.85), TBARS (r = 0.94), and NT (r = 0.99).
Cytokine/Chemokine markers of inflammation
Serum markers of inflammation [(interleukin-1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF)] were measured by using a commercially available Milliplex MAP 13-Plex Human High Sensitivity T-Cell Magnetic Bead Panel kit (EMD Millipore Corporation, St. Charles, MO, USA). A minimum of 100 positive beads for each cytokine/chemokine was acquired with a Luminex MagPix instrument (Luminex Corporation, Austin, TX, USA). Samples were run in duplicate according to standard procedures. Test to test variability of performing these assays yielded an average CV value range of ±4.26 to ±6.05 % for the aforementioned markers with an average test retest correlation of r = 0.99 for the same markers.
Statistical analysis
Individual group and time data are presented throughout as means (± SD), while group effects are presented as means (± SEM). All related variables were grouped and analyzed using repeated measures MANOVA in IBM SPSS Statistics Software version 22.0 for Windows (IBM Corporation, Armonk, NY, USA). Half-marathon finish time was also used as a covariate in subsequent ANCOVA analyses to determine if previously reported statistical outcomes were attributed to running intensity or to supplementation. Post-hoc LSD pairwise comparisons were used to analyze any significance among groups where needed with Cohen’s d calculations employed to determine effect magnitude. Data were considered statistically significant when the probability of error was less than 0.05 and considered to be trending when the probability of error was between 0.05 and 0.10.