Subjects and screening
Subjects were recruited from the Memphis area through the use of flyers placed on and around the University of Memphis campus. Eligibility was assessed via questionnaire. Our prior investigation of a 21-day Daniel Fast indicated that changes in outcome measures in response to the dietary plan were unaffected by gender, baseline body weight, or baseline exercise habits . Therefore, we elected to not place any restrictions on these variables regarding enrollment into the present study. Key inclusion criteria were men and women between the ages of 19–65 years with a body mass index (BMI) between 18–37 kg/m2. Key exclusion criteria were smoking, contraindication to krill oil or coconut oil (placebo) supplementation, pregnancy or desire to become pregnant, and unwillingness to follow a vegan diet. As these exclusion criteria were included in the recruitment flyers, outside of self-exclusion, none of the individuals that expressed interest in the study had to be excluded from participating. Sample size for this study was calculated based on the changes in total cholesterol and LDL-C that were observed in our previous investigation of a 21-day Daniel Fast . Forty-one subjects (13 men and 28 women) were enrolled, but 2 subjects (1 man and 1 woman) withdrew during the intervention phase due to personal reasons; therefore, 39 subjects were included in the analysis. Twenty-two subjects were classified as normal weight (BMI < 25 kg/m2), 9 were classified as overweight (BMI 25–29.9 kg/m2), and 8 were classified as obese (BMI ≥ 30 kg/m2). Thirty-three subjects were classified as exercise-trained, which was defined as performing ≥ 2 h/week of combined anaerobic and aerobic exercise of moderate to high intensity. On average, subjects performed 2.1 ± 0.2 h/week of anaerobic exercise for 6.8 ± 1.1 years and 4.7 ± 0.4 h/week of aerobic exercise for 8.3 ± 1.0 years. Six subjects had elevated BP (≥ 140/90 mm Hg), 7 subjects had elevated total cholesterol (≥ 200 mg/dL), and 2 subjects were type 2 diabetics. The following medication usages likely affected some of the outcome measurements in this study: Two subjects were taking hormone replacements, 2 were taking hypothyroid medication, 1 was taking an anti-inflammatory, 1 was taking insulin, 1 was taking hyperthyroid medication, and 1 was taking a stimulant/appetite suppressant. Failure to exclude individuals taking these medications during the screening process can be considered a limitation of this study. Subjects were instructed to not consume antioxidant supplements from 2 weeks prior to beginning the dietary plan until end of study. Prior to participation, each subject was informed of all procedures, potential risks, and benefits associated with the study in both verbal and written form in accordance with the approved procedures of the University Institutional Review Board for Human Subjects Research (protocol H11-14). Subjects provided written informed consent prior to being enrolled in the study.
The dietary intervention consisted of only consuming foods that comply with a Daniel Fast for 21 days. The 21-day duration was chosen to maximize generalizability – when motivated by religious reasons, most individuals follow a Daniel Fast dietary plan for 21 days, although 10- and 40-day Daniel Fasts have also been observed – and also to allow for a comparison against the findings from our previous study of the Daniel Fast . No meals were provided, and energy intake was unrestricted. Subjects were permitted to consume any food or beverage so long as it contained no animal products, white flour, preservatives, additives, sweeteners, flavorings, caffeine, or alcohol. Special emphasis was made to instruct the subjects to check the ingredients label of every food or beverage they planned to consume to ensure that it completely lacked any of the proscribed ingredients mentioned above. As an additional precaution, subjects were provided a list of commonly consumed foods that comply with the Daniel Fast as well as a second list of commonly consumed foods that do not. Subjects were also provided a basic recipe guide that only incorporated ingredients that comply with the Daniel Fast.
The outcome variables described below were measured immediately before (day 1) and after following the Daniel Fast dietary plan (day 22). All data collection occurred between 05:00 and 11:00 hours while subjects were in a 12-hour post-absorptive state.
Subjects were randomly assigned to consume one of the following for the duration of the Daniel Fast: Twenty-one subjects (7 men and 14 women) were assigned to orally consume krill oil capsules (NOW Foods, Bloomingdale, IL: 2 g/day in 2 daily dosages of 1 g), and 20 subjects (7 men and 13 women) were assigned to orally consume placebo capsules (coconut oil; NOW Foods, Bloomingdale, IL: 2 g/day in 2 daily dosages of 1 g). Specifically, men and women were alternately assigned to the krill oil group and the placebo group as they were enrolled in the study. Both the subjects and the investigators were blind to treatment via the use of coded bottles. A recent investigation examining the safety and tolerability of krill oil supplementation at 2 g/day for 4 weeks indicated that “krill oil was generally well tolerated and did not show evidence of any adverse influence on safety parameters” . Both of the subjects that withdrew from the study were in the krill oil group; thus, 19 subjects (6 men and 13 women) in the krill oil group completed the intervention. Subjects were instructed to consume 1 capsule in the morning with breakfast and another capsule in the evening with dinner, daily. Capsule counts upon bottle return at the end of the intervention were used to determine compliance to the supplementation. Each capsule of krill oil provided 230–300 mg of Omega-3 fatty acids, 140–160 mg of EPA, 80–90 mg of DHA, 10–20 mg of Omega-6 fatty acids, 80–90 mg of Omega-9 fatty acids, 390–420 mg of phospholipids, and 1.0-1.5 mg of esterified astaxanthin.
Blood pressure and heart rate
Subjects were seated in a chair with a BP cuff placed on their left arm. After a 10-minute period of quiet rest, two technicians measured heart rate by palpating the radial artery for 60 seconds. Blood pressure was then measured via auscultation using a dual-earpiece stethoscope that allowed for two technicians to listen simultaneously. Duplicate measures were obtained for both heart rate and BP, and the average of all measures was used in data analysis. If values deviated by more than 5 beats/minute for heart rate or 5 mm Hg for BP, an additional measure was taken.
Subjects’ height was measured using a stadiometer, and bodyweight was measured using a calibrated medical scale. Waist and hip circumference measurements were obtained using a tension-regulated measuring tape; subjects wore athletic shorts for these measurements. Fat mass, fat-free mass, total body fat percentage, and trunk-specific body fat percentage were determined via dual energy x-ray absorptiometry (Hologic QDR-4500 W) using a 4-min fan array.
Blood collection and biochemical variables
Blood samples that were collected in tubes containing EDTA were immediately separated via centrifugation at 1500 g for 15 minutes at 4°C for collection of plasma. Blood samples that were collected in tubes containing no additives were allowed to clot at room temperature for 30 minutes and then separated by centrifugation at 1500 g for 15 minutes at 4°C for collection of serum. A portion of blood samples were analyzed within 1 day of collection as follows: A complete blood count was performed using an automated cell counter (Coulter LH750). A comprehensive metabolic panel was obtained using automated procedures (Roche/Hitachi Modular). A lipid panel was obtained using enzymatic procedures (Roche/Hitachi Modular). C-reactive protein was measured using a high-sensitivity, particle-enhanced turbidimetric immunoassay (Roche Integra 800). Insulin was measured using an immuno-chemiluminescent assay procedure (Roche Modular E170). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as: fasting glucose (mg/dL) × fasting insulin (μU/mL)/405 .
The remaining blood samples were immediately stored in multiple aliquots at −70°C until analysis for the following: Malondialdehyde was analyzed in plasma following the procedures of Jentzsch et al.  using reagents purchased from Northwest Life Science Specialties (Vancouver, WA). Hydrogen peroxide was analyzed in plasma using the Amplex Red reagent method as described by the manufacturer (Molecular Probes, Invitrogen Detection Technologies, Eugene, OR). NOx was analyzed in plasma using a commercially available colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) according to the procedures provided by the manufacturer. Antioxidant capacity was analyzed in serum using the Trolox Equivalent Antioxidant Capacity assay according to the procedures outlined by the reagent provider (Sigma Chemical, St. Louis, MO). Resistin and adiponectin were analyzed in serum using an enzyme immunoassay according to the procedures provided by the manufacturer (SPI-Bio, Berin Pharma, France).
Dietary records and physical activity
All subjects were instructed to maintain their normal diet until they began the Daniel Fast. During the 7 days immediately prior to beginning the Daniel Fast, subjects recorded all food and beverages consumed. Subjects also recorded all food and beverages consumed during the final 7 days of the Daniel Fast. These dietary records were analyzed using Food Processor SQL, version 9.9 (ESHA Research, Salem, OR). Subjects were instructed to maintain their normal physical activity and exercise habits during the entire study period with one exception: Subjects were instructed to refrain from strenuous exercise during the 48 hours immediately preceding the 2 assessment days. Subjects were also instructed to refrain from alcohol consumption during the Daniel Fast (as detailed in the dietary guidelines) and also during the 48 hours that preceded day 1.
Compliance to food intake, rating of physical and mental health, and satiety
At the conclusion of the intervention period (day 22), subjects completed a questionnaire pertaining to their experience with the Daniel Fast. Using a scale of 1–10 (1 = as low as possible; 10 = as high as possible), subjects rated their feeling of physical health and vitality, their mental health (overall outlook and mood), and their level of satiety while following the dietary plan. Subjects also rated their compliance to the Daniel Fast using a scale of 0–100 (0 = complete noncompliance; 100 = complete compliance).
All outcome measures were analyzed using a 2 (condition) × 2 (pre/post intervention) ANOVA. However, data for both conditions were subsequently collapsed and analyzed using a paired t-test. Analyses were performed using JMP statistical software (version 4.0.3, SAS Institute, Cary, NC). Statistical significance was set at p ≤ 0.05.