# The Effects of Krill Oil on mTOR Signaling and Resistance[tnq_nbsp] Exercise: A Pilot Study

Authors: John Georges; Matthew H. Sharp; Ryan P. Lowery; Jacob M. Wilson; Martin Purpura; Troy A. Hornberger; Flint Harding; James H. Johnson; David M. Peele; Ralf Jäger
Journal: Journal of Nutrition and Metabolism
 (2018)

---

## Abstract

Introduction. Krill oil supplementation has been shown to improve postexercise immune function; however, its effect on muscle hypertrophy is currently unknown. Therefore, the aim of present study was to investigate the ability of krill oil to stimulate mTOR signaling and its ability to augment resistance 3b2:?thyc=10training-induced3b2:?thyc changes in body composition and performance. Methods. C2C12 myoblasts cells were stimulated with krill oil or 3b2:?thyc=10soy-derived3b2:?thyc phosphatidylcholine (3b2:?thyc=10S-PC),3b2:?thyc and then, the ratio of P-3b2:?show $132#3b2:?thyc=10p70-3893b2:?thyc to total p70 was used as readout for mTOR signaling. In 3b2:?thyc=10double-blind,3b2:?thyc 3b2:?thyc=10placebo-controlled3b2:?thyc study, resistance trained subjects consumed either 3 g krill oil daily or placebo, and each took part in an 8-week periodized resistance training program. Body composition, maximal strength, peak power, and rate of perceived recovery were assessed collectively at the end of weeks 0 and 8. In addition, safety parameters (comprehensive metabolic panel (CMP), complete blood count (CBC), and urine analysis (UA)) and cognitive performance were measured pre- and posttesting. Results. Krill oil significantly stimulated mTOR signaling in comparison to S-3b2:?show $132#PC and control. No differences for markers on the CMP, CBC, or UA were observed. Krill oil significantly increased lean body mass from baseline (p=0.021, 1.4 kg, +2.1%); however, there were no statistically significant differences between groups for any measures taken. Conclusion. Krill oil activates mTOR signaling. Krill oil supplementation in athletes is safe, and its effect on resistance exercise deserves further research.


---

## Body

## 1. Introduction

3b2:?A3B2 tpt 1ptAthletes use nutrition strategies to improve their training and performance through increasing their metabolic capacity, delaying the onset of fatigue, and improving muscle hypertrophy by enhancing recovery, improving immune function, and decreasing oxidative stress. Krill oil is rich in 3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which have been found to have positive effects on inflammation [1]. In krill oil, 3b2:?thyc=10omega-33b2:?thyc PUFAs are bound to[tnq_nbsp] phospholipids (PL), whereas in fish oil, the majority of 3b2:?thyc=10omega-33b2:?thyc PUFAs are bound to triacylglycerol (TG) [2]. Greater bioavailability of 3b2:?thyc=10omega-33b2:?thyc PUFA from krill oil in comparison to fish oil has been suggested based on lower doses of krill oil needed to result in a similar bloodstream level of EPA 3b2:?thyc=10and/or 3b2:?thycDHA; however, more carefully controlled human trials are needed to establish their relative efficacies after chronic administration [3]. Contrasting results have been found on the effects of fish oil supplementation on muscle damage and postexercise soreness in healthy men and women. While most of studies at ranges from 2.4 to 6 3b2:?thyc=10g/d 3b2:?thycfor 1 to 8 weeks have shown beneficial effects [4–3b2:?show $132#8], two studies showed no effect [9,[tnq_nbsp] 10], potentially due to different types of exercise used to induce muscle damage or differences in the dose and duration of 3b2:?thyc=10omega-33b2:?thyc PUFA supplementation among studies. A combination of EPA and DHA was found to increase the rates of muscle protein synthesis via an increase in activation of the 3b2:?thyc=10mTOR-p70s6k3b2:?thyc signaling pathway in young- and 3b2:?thyc=10middle-aged3b2:?thyc men and women [11]. Fish oil supplementation in combination with [12] or without [13] resistance exercise resulted in increased strength and functional ability in older adults. However, potential 3b2:?thyc=10long-term3b2:?thyc benefits of 3b2:?thyc=10omega-33b2:?thyc PUFA supplementation on muscle hypertrophy and performance in young healthy subjects undergoing a controlled resistance training program are currently unknown.3b2:?A3B2 tlsb=0.09ptKrill oil contains astaxanthin, a red carotenoid pigment and strong antioxidant that naturally occurs in salmon, shrimp, krill, crustaceans, or certain types of algae, giving krill its reddish color. Astaxanthin administration has been[tnq_nbsp] shown to reduce muscle damage [14,[tnq_nbsp] 15], to increase time trial performance and power output in competitive cyclists [16], and to increase 3b2:?thyc=10strength/endurance 3b2:?thyc(number of squats) [17]. However, astaxanthin failed to improve muscle soreness and muscle damage in resistance trained men following an acute bout of eccentric exercise (coadministered with lutein) [18]. Discrepancies in astaxanthin’s ability to attenuate 3b2:?thyc=10exercise-induced3b2:?thyc muscle injury might be due to the type of exercise stress (resistance or endurance exercise) or the dosage and timing of the administration. Krill oil contains approximately 0.5 mg of astaxanthin per 3 g of krill oil, which is below the currently established effective dose of 4 mg for athletes. However, the absorption of astaxanthin can be greatly enhanced in the presence of fats, surfactants, or phospholipids. The phospholipid content in krill oil will likely increase the absorption of astaxanthin closing the gap between the actual content and effective dose.In athletes, krill oil has been shown to improve postexercise immune function (23b2:?thyc=10g/d 3b2:?thycfor six weeks) [19] and diminished postexercise oxidative damage during recovery (1 3b2:?thyc=10g/d 3b2:?thycfor six weeks) [20]; however, it failed to improve exercise performance (cycling time trial [19] and total run time in a 2,000 m test [20]). The lack of performance benefits of krill oil supplementation in previous exercise studies might have been based on a lack of an accompanying controlled challenging training protocol optimizing krill oil’s benefits on recovery, as muscle recovery after an exercise bout might influence training adaptations. While krill oil’s effect on mTOR signaling is currently unknown, 3b2:?thyc=10DHA/EPA 3b2:?thychas been shown to activate the 3b2:?thyc=10mTOR-p70s6k3b2:?thyc signaling pathway [11]. A comparison of 3b2:?thyc=10soy-derived3b2:?thyc (containing no 3b2:?thyc=10omega-33b2:?thyc PUFAs) and 3b2:?thyc=10PUFA-containing3b2:?thyc 3b2:?thyc=10egg-derived3b2:?thyc phosphatidic acid showed a potential attenuating effect of PUFAs bound to PL on mTOR activation [21]. Therefore, the aim of the present study was to investigate the ability of krill oil to stimulate mTOR signaling and its ability to augment resistance 3b2:?thyc=10training-induced3b2:?thyc changes in body composition and performance.


## 2. Materials and Methods

### 2.1. Cell Culture Tests

C2C12 myoblasts (ATCC; Manassas, Virginia) were plated at approximately 30% confluence and grown for 24 hours in 10% FBS high glucose DMEM with antibiotics (100 μ3b2:?thyc=10g/ml 3b2:?thycstreptomycin and 100 3b2:?thyc=10U/ml 3b2:?thycpenicillin; Sigma). At 16 hours prior to the experiment, myoblasts cells were switched to 3b2:?thyc=10serum-free3b2:?thyc 3b2:?thyc=10high-glucose3b2:?thyc DMEM (no antibiotics) and were approximately 70% confluent at the time of the experiment with no indication of myoblast fusion present. All stimulants were dissolved in chloroform to yield a concentration of 10 3b2:?thyc=10mg/mL. 3b2:?thycEach stimulant was then dried 3b2:?A3B2 tlsb=0.12ptwith a stream of nitrogen gas and resuspended in PBS to obtain either 20 or 60 nmol/100 µL, such that 100 µL added to 2 mL of media resulted in 10 or 30 µM, respectively. Accordingly, cells were stimulated for 20 minutes with vehicle (Control; 100 µL of PBS), 10 or 30 µM of krill oil (Rimfrost USA, Merry Hill, North Carolina, USA) or S-3b2:?show $132#PC (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) as the negative control. Cells were[tnq_nbsp] then harvested in lysis buffer (40 mM Tris, pH 7.5; 1 mM[tnq_nbsp] EDTA; 5 mM EGTA; 0.5% Triton X-3b2:?show $132#100; 25 mM β-glycerophosphate; 25 mM NaF; 1 mM Na3VO4; 10 µ3b2:?thyc=10g/mL 3b2:?thycleupeptin; and 1 mM PMSF) and subjected to immunoblotting. Equivalent amounts of protein from each sample were dissolved in Laemmli buffer and subjected to electrophoretic separation on 7.5% 3b2:?thyc=10SDS-PAGE.3b2:?thyc Following electrophoretic separation, proteins were transferred to a polyvinylidene difluoride membrane in transfer buffer (242 mM Tris, 58 mM glycine). Membranes were blocked with 5% powdered milk in TBST (3b2:?thyc=10Tris-buffered3b2:?thyc saline with 0.1% Tween 20) for 1 h followed by an overnight incubation at 4°C with rabbit 3b2:?thyc=10anti-phospho-p703b2:?thyc S6 kinase (3b2:?thyc=10P-p70(389);3b2:?thyc Cell signaling #9234; 1 : 1000) dissolved in TBST containing 1% BSA. Membranes were washed for[tnq_nbsp] 30 min in TBST and then incubated for 1 h at[tnq_nbsp] room temperature in 5% 3b2:?thyc=10milk-TBST3b2:?thyc containing 3b2:?thyc=10peroxidase-conjugated3b2:?thyc 3b2:?thyc=10anti-rabbit3b2:?thyc (3b2:?thyc=10PI-10003b2:?thyc Vector Laboratories, Burlingame, CA, USA). After 30 min of washing in TBST, the blots were developed on a Chemi410 camera mounted to a UVP AutoChemi system (UVP, Upland, CA, USA) using 3b2:?thyc=10prime-enhanced3b2:?thyc chemiluminescence (ECL) reagent (Pierce; Thermo Fisher Scientific, Rockford, IL, USA). Once the appropriate image was captured, the membranes were stripped for 30 minutes in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM 3b2:?thyc=10Tris-HCl3b2:?thyc pH 6.8) maintained at 50°C. Membranes were washed with TBST and then reblocked with 5% powdered milk in TBST for 1 h followed by an overnight incubation at 4°C with rabbit 3b2:?thyc=10anti-p703b2:?thyc S6 kinase (total p70, cell signaling #2708, 1 : 2000) dissolved in TBST containing 1% BSA. Membranes were washed for 30 min in TBST and then incubated for 1 h at room temperature in 5% 3b2:?thyc=10milk-TBST3b2:?thyc containing 3b2:?thyc=10peroxidase-conjugated3b2:?thyc 3b2:?thyc=10anti-rabbit.3b2:?thyc After 30 min of washing in TBST, the blots were developed on UVP using a regular ECL reagent. Once the appropriate image was captured, the membranes were stained with Coomassie blue to verify equal loading in all lanes. Densitometric measurements were performed by determining the density of each band using the public domain ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/nih-image/). The ratio of P-3b2:?show $132#p70(389) to total p70 was used as a readout for mTOR signaling.3b2:?A3B2 tpb=3.5pt


### 2.2. Human Efficacy Study

#### 2.2.1. Study Design

This study consisted of a randomized,3b2:?thyc=10double-blind3b2:?thyc protocol consisting of 2 groups of individuals 3b2:?A3B2 tlsb=-0.15ptgiven either 3 g of placebo (olive oil) or 3 g of krill (Euphausia superba) oil (Rimfrost Sublime, Rimfrost USA, LLC, Merry Hill, NC, USA, Lot 8723-15-01-03, consisting of 43.8% phospholipids (1.3 g), delivering 963 mg total 3b2:?thyc=10omega-33b2:?thyc fatty acids (240 mg DHA, 393 mg EPA), and 0.54 mg astaxanthin) 3b2:?thyc=10pre-workout3b2:?thyc on training days, and with breakfast on nontraining days, during an 8-week high intensity, nonlinear, periodized resistance training protocol in form of 6 500 mg softgel capsules. The softgel capsules were manufactured by Aenova, Miami, FL, USA. Body composition, maximal strength, peak power, and rate of perceived recovery were assessed collectively at the end of weeks 0 and 8. In addition, safety parameters (comprehensive metabolic panel (CMP), complete blood count (CBC), and urine analysis (UA)) were measured pre- and posttesting.


#### 2.2.2. Subjects

Forty subjects were assessed for eligibility and of the3b2:?thyc=10twenty-one3b2:?thyc subjects enrolled 11 subjects received the placebo and 10 received krill oil. A total of 3 subjects were lost to 3b2:?thyc=10follow-up3b2:?thyc by a lack of communication with the researcher (2 from the placebo and 1 from the krill oil group). A total of 18 subjects were analyzed, 9 from each condition (Figure 3b2:?show [?"gr1",5,"cond1"]1).Figure 1
CONSORT chart.3b2:?A3B2 tlsb=-0.29ptSubject inclusion criteria were males 18 to 30 years of age, resistance training at least 2 times per week for the past six months, a minimum of 1 year of training experience active and currently resistance training, free of musculoskeletal, metabolic, and respiratory disorders, free of cardiovascular disease, no musculoskeletal injuries with the last six months, no history of smoking or drug use, no history of excessive alcohol consumption, not taking prescription medication, have not used a fish oil-, thermogenic-, protein-, amino acid-, or creatine supplement within the prior two months, and have not habitually used caffeine (3b2:?thyc=10e.3b2:?show $132#g.,3b2:?thyc[tnq_nbsp] more than 2 cups of coffee per day). Subjects were 3b2:?thyc=10matched-paired3b2:?thyc by age, body mass, strength and resistance training, and physical activity background and then randomly placed into one of the two groups. Subjects were prohibited from consuming any nutritional supplements (including fish oil) for the duration of the study. Additionally, subjects were instructed to avoid consumption of all fish and fish byproducts. After an explanation of the procedures and associated risks, all volunteers completed written informed consent. All procedures were approved by the IntegReview Institutional Review Board, Austin, TX, USA (protocol #7952). This study was registered with the ISRCTN registry (ISRCTN11524409). Subject characteristic data are displayed in Table 3b2:?show [?"tbl1",5,"cond1"]1.Table 1
Subject characteristic data.
Age (yrs)
Training age (yrs)
Body mass (kg)
Height (cm)
3b2:?show "^tbl_width"$01649v
Placebo(n=9)
22.0 ± 0.6
3.0 ± 0.2
79.9 ± 3.9
179.3 ± 3.1
Krill oil(n=9)
22.3 ± 0.4
3.1 ± 0.7
82.9 ± 5.0
178.8 ± 2.3


#### 2.2.3. Resistance Exercise Training Protocol

Resistance training occurred four days per week (programmed, nonlinear training split). The resistance training protocol was modified from Kraemer et[tnq_nbsp] al. [22] and Montiero et[tnq_nbsp] al. [23]. These researchers found that a nonlinear resistance training program yielded greater results than a traditional or nonperiodized program in athletes (Montiero et[tnq_nbsp] al. [23]). The program was designed to train all major muscle groups using many compound movements for the upper body (3b2:?thyc=10e.3b2:?show $132#g.,3b2:?thyc[tnq_nbsp] bench press, dips, shoulder press, 3b2:?thyc=10pull-ups,3b2:?thyc and bent over rows), lower body (leg press, leg extensions, and 3b2:?thyc=10glute-ham3b2:?thyc raises), and the core. The programmed, nonlinear training split was divided into hypertrophy days consisting of 8–3b2:?show $132#10 RM loads for 3 sets, with 90 seconds rest, strength endurance days consisting of 12–3b2:?show $132#15 repetitions, with 60 seconds rest, and on days training maximal strength consisting of 3 to 5 RM loads with 3 sets for all exercises except the leg press and bench press (5 total sets). Weights were progressively increased or decreased on a 3b2:?thyc=10set-by-set3b2:?thyc basis by 2–3b2:?show $132#5% to meet prescribed repetitions. Briefly, when subjects could have achieved more[tnq_nbsp] than 2 repetitions over the prescribed number, the load[tnq_nbsp] was 3b2:?A3B2 tlsb .2ptincreased by 2–3b2:?show $132#5%. When subjects failed to reach the prescribed repetitions under their own will, the load was decreased by 2–3b2:?show $132#5%. All training sessions were closely monitored to ensure effort and intensity to maximal on each training sessions. Subjects trained each body 3b2:?A3B2 tlsb=-0.25ptpart twice weekly and alternately between hypertrophy and heavy 3b2:?A3B2 tlsb .25ptworkouts. We have selected this protocol because a 3b2:?thyc=10meta-3b2:?show $132#analysis3b2:?thyc by Rhea [24] indicated that training at this frequency was ideal for moderately resistance trained individuals (Table 2).Table 2
Training Protocol.
Weekly Schedule
Weight Training
Week
1E
2H
3E
4H
Hypertrophy/endurance:
3 sets/12 repetitions60s rest
3 sets/8 repetitions90 s rest
3 sets/15 repetitions60s rest
3 sets/10 repetitions90s rest
Strength:
3–5 sets/5 repetitions3–5 min rest
3–5 sets/4 repetitions3–5 min rest
3–5 sets/3 repetitions3–5 min rest
3–5 sets/5 repetitions3–5 min rest
Week
5
E
6
H
7
E
8
H
Hypertrophy/endurance:
3 sets/12 repetitions90 s rest
3 sets/8 repetitions90s rest
3 sets/15 repetitions60s rest
3 sets/10 repetitions90s rest
Strength:
3–5 sets/ 4 repetitions3–5 min rest
3–5 sets/4 repetitions3–5 min rest
3–5 sets/ 3 repetitions3–5 min rest
3–5 sets/ 3 repetitions3–5 min rest
Exercise Selection
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
Strength
Rest
Hypertrophy/endurance
Hypertrophy/endurance
Rest
Strength
Leg press
Bench press
Squat
Bench press
Squat
BB row
Deadlift
BB row
3b2:?A3B2 tlsb -0.25ptGlute-ham raise
BB shoulder press
Leg press
BB shoulder press
Pull-ups
Glute-ham raise
Pull-ups
DB press
Seated leg curl
Incline DB press
Lying leg curl
Side lateral raise
Leg ext.
Preacher curl
Calf raise
Overhead tricep ext.
Cable fly
Hammer curl
Tricep pushdown
Cable press


#### 2.2.4. Body Composition

3b2:?A3B2 tlsb 0.1ptA 3b2:?thyc=10whole-body3b2:?thyc 3b2:?thyc=10dual-energy3b2:?thyc X-3b2:?show $132#ray absorptiometry (DXA) (Hologic, Bedford, MA, USA) scan was utilized to measure body composition. Lean body mass (LBM) and fat mass (FM) were determined for the total body with the subject laying in a supine position with the knee extended and instructed not to move for the entire duration of the scan (∼5 minutes). Results from each scan were uploaded and accessed on a computer directly connected to the DXA device. All DXA scans were conducted prior to and after the completion of the study, and each subject was required to fast overnight (10 hours) prior to the DXA scan. Calibration of the DXA device was done against a phantom provided by the manufacturing company prior to testing.


#### 2.2.5.3b2:?A3B2 tlsb -.21ptStrength Assessment

3b2:?thyc=10One-repetition3b2:?thyc maximum (1 RM) 3b2:?show [?tjl=20mm]&tjlpc;[?tjl]was assessed on bench press and leg press at baseline and after 8 weeks. Loads were increased incrementally until maximal load or failure at a given load was reached. Briefly, subjects performed a general warmup and a specific warmup consisting of three sets. During the first set, subjects performed 10 repetitions with 50% of their predicted 1 RM. For the second set, they performed five repetitions with 70% of the predicted 1 RM. In the third set, subjects perform one repetition with 90% of their predicted 1 RM. After the completion of warmup sets, subjects rested for 3 minutes. Then, each subject had as many as five attempts to achieve their 1 RM load with 3–3b2:?show $132#5 minutes rest between each attempt.


#### 2.2.6. Perceptual Measures

The perceptual measures were collected using a perceived recovery status scale. Ratings of perceived recovery were collected at the beginning and end of[tnq_nbsp] every week. The perceived recovery status scale consisted of a scalar representation numbering from 0 to 10. Visual descriptors of “very poorly recovered,” “adequately recovered,” and “very well recovered” for perceived recovery were presented at numbers 0, 5, and 10, respectively. Subjects were asked to identify their level of perceived recovery after warming up and before performing the training protocol.


#### 2.2.7. Stroop Test

The Stroop test is a psychological test of mental vitality and flexibility. The test consists of computerized presentation of the names of four colors (yellow, blue, green, and red), written in capital letters, Times New Roman font, size 24, in black. The order is semirandom, so that the same word never appears two consecutive times throughout the test. The subject’s task is to read each word as quickly as possible. This part of the test is intended to obtain a baseline to evaluate the reading ability and determine whether this ability is high enough so as not to hinder the interference effect. This is because the effect of3b2:?thyc=10color-word3b2:?thyc interference may be absent if the reading ability is lower than expected. Characterized by a series of colorized words, which themselves are colors, subjects were instructed to read the color of the word aloud and not the word itself. Correct answers and time to completion were recorded. “Congruent” indicates the number of correct responses out of five responses to the test with matching words and colors. “Incongruent” indicates the number of correct responses out of fifteen responses to the test with nonmatched words and colors.


#### 2.2.8. Blood Safety Markers

Subjects donated approximately 10 mL of fasted, whole blood at the baseline and at the end of week 8. All blood samples were collected by a trained phlebotomist via venipuncture of an antecubital vein in the3b2:?A3B2 tlsb=-0.15ptforearm using standard sterile procedures. Biomarkers comprising CMP, CBC, and lipid panels were assayed for data collection.


#### 2.2.9. Statistical Analysis

3b2:?A3B2 tlsb=-0.3ptBefore carrying out the parametric statistical analysis, dependent variables were examined for a normal distribution and outliers through investigation of boxplots and a normality test (3b2:?thyc=10i.3b2:?show $132#e.,3b2:?thyc[tnq_nbsp] Shapiro–3b2:?show $132#Wilk). Normality tests revealed no outliers, and analysis was performed using the original data set. Repeated measures ANOVA were used to scrutinize the effects of supplementation on dependent variables assuming the group (placebo and krill oil) and time (pre- and posttest) as fixed factors (GraphPad Prism 7®, La Jolla, CA). Whenever an interaction or main effect was demonstrated, a Bonferroni post hoc test was performed to identify where the differences occurred. The significance level was previously set at p<0.05. All data are reported as mean ± SEM. Analysis of baseline characteristic revealed that there were no statistical significant difference between groups for the investigated dependent variables (p>0.05).


## 2.1. Cell Culture Tests

C2C12 myoblasts (ATCC; Manassas, Virginia) were plated at approximately 30% confluence and grown for 24 hours in 10% FBS high glucose DMEM with antibiotics (100 μ3b2:?thyc=10g/ml 3b2:?thycstreptomycin and 100 3b2:?thyc=10U/ml 3b2:?thycpenicillin; Sigma). At 16 hours prior to the experiment, myoblasts cells were switched to 3b2:?thyc=10serum-free3b2:?thyc 3b2:?thyc=10high-glucose3b2:?thyc DMEM (no antibiotics) and were approximately 70% confluent at the time of the experiment with no indication of myoblast fusion present. All stimulants were dissolved in chloroform to yield a concentration of 10 3b2:?thyc=10mg/mL. 3b2:?thycEach stimulant was then dried 3b2:?A3B2 tlsb=0.12ptwith a stream of nitrogen gas and resuspended in PBS to obtain either 20 or 60 nmol/100 µL, such that 100 µL added to 2 mL of media resulted in 10 or 30 µM, respectively. Accordingly, cells were stimulated for 20 minutes with vehicle (Control; 100 µL of PBS), 10 or 30 µM of krill oil (Rimfrost USA, Merry Hill, North Carolina, USA) or S-3b2:?show $132#PC (Avanti Polar Lipids, Inc., Alabaster, Alabama, USA) as the negative control. Cells were[tnq_nbsp] then harvested in lysis buffer (40 mM Tris, pH 7.5; 1 mM[tnq_nbsp] EDTA; 5 mM EGTA; 0.5% Triton X-3b2:?show $132#100; 25 mM β-glycerophosphate; 25 mM NaF; 1 mM Na3VO4; 10 µ3b2:?thyc=10g/mL 3b2:?thycleupeptin; and 1 mM PMSF) and subjected to immunoblotting. Equivalent amounts of protein from each sample were dissolved in Laemmli buffer and subjected to electrophoretic separation on 7.5% 3b2:?thyc=10SDS-PAGE.3b2:?thyc Following electrophoretic separation, proteins were transferred to a polyvinylidene difluoride membrane in transfer buffer (242 mM Tris, 58 mM glycine). Membranes were blocked with 5% powdered milk in TBST (3b2:?thyc=10Tris-buffered3b2:?thyc saline with 0.1% Tween 20) for 1 h followed by an overnight incubation at 4°C with rabbit 3b2:?thyc=10anti-phospho-p703b2:?thyc S6 kinase (3b2:?thyc=10P-p70(389);3b2:?thyc Cell signaling #9234; 1 : 1000) dissolved in TBST containing 1% BSA. Membranes were washed for[tnq_nbsp] 30 min in TBST and then incubated for 1 h at[tnq_nbsp] room temperature in 5% 3b2:?thyc=10milk-TBST3b2:?thyc containing 3b2:?thyc=10peroxidase-conjugated3b2:?thyc 3b2:?thyc=10anti-rabbit3b2:?thyc (3b2:?thyc=10PI-10003b2:?thyc Vector Laboratories, Burlingame, CA, USA). After 30 min of washing in TBST, the blots were developed on a Chemi410 camera mounted to a UVP AutoChemi system (UVP, Upland, CA, USA) using 3b2:?thyc=10prime-enhanced3b2:?thyc chemiluminescence (ECL) reagent (Pierce; Thermo Fisher Scientific, Rockford, IL, USA). Once the appropriate image was captured, the membranes were stripped for 30 minutes in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM 3b2:?thyc=10Tris-HCl3b2:?thyc pH 6.8) maintained at 50°C. Membranes were washed with TBST and then reblocked with 5% powdered milk in TBST for 1 h followed by an overnight incubation at 4°C with rabbit 3b2:?thyc=10anti-p703b2:?thyc S6 kinase (total p70, cell signaling #2708, 1 : 2000) dissolved in TBST containing 1% BSA. Membranes were washed for 30 min in TBST and then incubated for 1 h at room temperature in 5% 3b2:?thyc=10milk-TBST3b2:?thyc containing 3b2:?thyc=10peroxidase-conjugated3b2:?thyc 3b2:?thyc=10anti-rabbit.3b2:?thyc After 30 min of washing in TBST, the blots were developed on UVP using a regular ECL reagent. Once the appropriate image was captured, the membranes were stained with Coomassie blue to verify equal loading in all lanes. Densitometric measurements were performed by determining the density of each band using the public domain ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA; http://rsb.info.nih.gov/nih-image/). The ratio of P-3b2:?show $132#p70(389) to total p70 was used as a readout for mTOR signaling.3b2:?A3B2 tpb=3.5pt


## 2.2. Human Efficacy Study

### 2.2.1. Study Design

This study consisted of a randomized,3b2:?thyc=10double-blind3b2:?thyc protocol consisting of 2 groups of individuals 3b2:?A3B2 tlsb=-0.15ptgiven either 3 g of placebo (olive oil) or 3 g of krill (Euphausia superba) oil (Rimfrost Sublime, Rimfrost USA, LLC, Merry Hill, NC, USA, Lot 8723-15-01-03, consisting of 43.8% phospholipids (1.3 g), delivering 963 mg total 3b2:?thyc=10omega-33b2:?thyc fatty acids (240 mg DHA, 393 mg EPA), and 0.54 mg astaxanthin) 3b2:?thyc=10pre-workout3b2:?thyc on training days, and with breakfast on nontraining days, during an 8-week high intensity, nonlinear, periodized resistance training protocol in form of 6 500 mg softgel capsules. The softgel capsules were manufactured by Aenova, Miami, FL, USA. Body composition, maximal strength, peak power, and rate of perceived recovery were assessed collectively at the end of weeks 0 and 8. In addition, safety parameters (comprehensive metabolic panel (CMP), complete blood count (CBC), and urine analysis (UA)) were measured pre- and posttesting.


### 2.2.2. Subjects

Forty subjects were assessed for eligibility and of the3b2:?thyc=10twenty-one3b2:?thyc subjects enrolled 11 subjects received the placebo and 10 received krill oil. A total of 3 subjects were lost to 3b2:?thyc=10follow-up3b2:?thyc by a lack of communication with the researcher (2 from the placebo and 1 from the krill oil group). A total of 18 subjects were analyzed, 9 from each condition (Figure 3b2:?show [?"gr1",5,"cond1"]1).Figure 1
CONSORT chart.3b2:?A3B2 tlsb=-0.29ptSubject inclusion criteria were males 18 to 30 years of age, resistance training at least 2 times per week for the past six months, a minimum of 1 year of training experience active and currently resistance training, free of musculoskeletal, metabolic, and respiratory disorders, free of cardiovascular disease, no musculoskeletal injuries with the last six months, no history of smoking or drug use, no history of excessive alcohol consumption, not taking prescription medication, have not used a fish oil-, thermogenic-, protein-, amino acid-, or creatine supplement within the prior two months, and have not habitually used caffeine (3b2:?thyc=10e.3b2:?show $132#g.,3b2:?thyc[tnq_nbsp] more than 2 cups of coffee per day). Subjects were 3b2:?thyc=10matched-paired3b2:?thyc by age, body mass, strength and resistance training, and physical activity background and then randomly placed into one of the two groups. Subjects were prohibited from consuming any nutritional supplements (including fish oil) for the duration of the study. Additionally, subjects were instructed to avoid consumption of all fish and fish byproducts. After an explanation of the procedures and associated risks, all volunteers completed written informed consent. All procedures were approved by the IntegReview Institutional Review Board, Austin, TX, USA (protocol #7952). This study was registered with the ISRCTN registry (ISRCTN11524409). Subject characteristic data are displayed in Table 3b2:?show [?"tbl1",5,"cond1"]1.Table 1
Subject characteristic data.
Age (yrs)
Training age (yrs)
Body mass (kg)
Height (cm)
3b2:?show "^tbl_width"$01649v
Placebo(n=9)
22.0 ± 0.6
3.0 ± 0.2
79.9 ± 3.9
179.3 ± 3.1
Krill oil(n=9)
22.3 ± 0.4
3.1 ± 0.7
82.9 ± 5.0
178.8 ± 2.3


### 2.2.3. Resistance Exercise Training Protocol

Resistance training occurred four days per week (programmed, nonlinear training split). The resistance training protocol was modified from Kraemer et[tnq_nbsp] al. [22] and Montiero et[tnq_nbsp] al. [23]. These researchers found that a nonlinear resistance training program yielded greater results than a traditional or nonperiodized program in athletes (Montiero et[tnq_nbsp] al. [23]). The program was designed to train all major muscle groups using many compound movements for the upper body (3b2:?thyc=10e.3b2:?show $132#g.,3b2:?thyc[tnq_nbsp] bench press, dips, shoulder press, 3b2:?thyc=10pull-ups,3b2:?thyc and bent over rows), lower body (leg press, leg extensions, and 3b2:?thyc=10glute-ham3b2:?thyc raises), and the core. The programmed, nonlinear training split was divided into hypertrophy days consisting of 8–3b2:?show $132#10 RM loads for 3 sets, with 90 seconds rest, strength endurance days consisting of 12–3b2:?show $132#15 repetitions, with 60 seconds rest, and on days training maximal strength consisting of 3 to 5 RM loads with 3 sets for all exercises except the leg press and bench press (5 total sets). Weights were progressively increased or decreased on a 3b2:?thyc=10set-by-set3b2:?thyc basis by 2–3b2:?show $132#5% to meet prescribed repetitions. Briefly, when subjects could have achieved more[tnq_nbsp] than 2 repetitions over the prescribed number, the load[tnq_nbsp] was 3b2:?A3B2 tlsb .2ptincreased by 2–3b2:?show $132#5%. When subjects failed to reach the prescribed repetitions under their own will, the load was decreased by 2–3b2:?show $132#5%. All training sessions were closely monitored to ensure effort and intensity to maximal on each training sessions. Subjects trained each body 3b2:?A3B2 tlsb=-0.25ptpart twice weekly and alternately between hypertrophy and heavy 3b2:?A3B2 tlsb .25ptworkouts. We have selected this protocol because a 3b2:?thyc=10meta-3b2:?show $132#analysis3b2:?thyc by Rhea [24] indicated that training at this frequency was ideal for moderately resistance trained individuals (Table 2).Table 2
Training Protocol.
Weekly Schedule
Weight Training
Week
1E
2H
3E
4H
Hypertrophy/endurance:
3 sets/12 repetitions60s rest
3 sets/8 repetitions90 s rest
3 sets/15 repetitions60s rest
3 sets/10 repetitions90s rest
Strength:
3–5 sets/5 repetitions3–5 min rest
3–5 sets/4 repetitions3–5 min rest
3–5 sets/3 repetitions3–5 min rest
3–5 sets/5 repetitions3–5 min rest
Week
5
E
6
H
7
E
8
H
Hypertrophy/endurance:
3 sets/12 repetitions90 s rest
3 sets/8 repetitions90s rest
3 sets/15 repetitions60s rest
3 sets/10 repetitions90s rest
Strength:
3–5 sets/ 4 repetitions3–5 min rest
3–5 sets/4 repetitions3–5 min rest
3–5 sets/ 3 repetitions3–5 min rest
3–5 sets/ 3 repetitions3–5 min rest
Exercise Selection
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
Strength
Rest
Hypertrophy/endurance
Hypertrophy/endurance
Rest
Strength
Leg press
Bench press
Squat
Bench press
Squat
BB row
Deadlift
BB row
3b2:?A3B2 tlsb -0.25ptGlute-ham raise
BB shoulder press
Leg press
BB shoulder press
Pull-ups
Glute-ham raise
Pull-ups
DB press
Seated leg curl
Incline DB press
Lying leg curl
Side lateral raise
Leg ext.
Preacher curl
Calf raise
Overhead tricep ext.
Cable fly
Hammer curl
Tricep pushdown
Cable press


### 2.2.4. Body Composition

3b2:?A3B2 tlsb 0.1ptA 3b2:?thyc=10whole-body3b2:?thyc 3b2:?thyc=10dual-energy3b2:?thyc X-3b2:?show $132#ray absorptiometry (DXA) (Hologic, Bedford, MA, USA) scan was utilized to measure body composition. Lean body mass (LBM) and fat mass (FM) were determined for the total body with the subject laying in a supine position with the knee extended and instructed not to move for the entire duration of the scan (∼5 minutes). Results from each scan were uploaded and accessed on a computer directly connected to the DXA device. All DXA scans were conducted prior to and after the completion of the study, and each subject was required to fast overnight (10 hours) prior to the DXA scan. Calibration of the DXA device was done against a phantom provided by the manufacturing company prior to testing.


### 2.2.5.3b2:?A3B2 tlsb -.21ptStrength Assessment

3b2:?thyc=10One-repetition3b2:?thyc maximum (1 RM) 3b2:?show [?tjl=20mm]&tjlpc;[?tjl]was assessed on bench press and leg press at baseline and after 8 weeks. Loads were increased incrementally until maximal load or failure at a given load was reached. Briefly, subjects performed a general warmup and a specific warmup consisting of three sets. During the first set, subjects performed 10 repetitions with 50% of their predicted 1 RM. For the second set, they performed five repetitions with 70% of the predicted 1 RM. In the third set, subjects perform one repetition with 90% of their predicted 1 RM. After the completion of warmup sets, subjects rested for 3 minutes. Then, each subject had as many as five attempts to achieve their 1 RM load with 3–3b2:?show $132#5 minutes rest between each attempt.


### 2.2.6. Perceptual Measures

The perceptual measures were collected using a perceived recovery status scale. Ratings of perceived recovery were collected at the beginning and end of[tnq_nbsp] every week. The perceived recovery status scale consisted of a scalar representation numbering from 0 to 10. Visual descriptors of “very poorly recovered,” “adequately recovered,” and “very well recovered” for perceived recovery were presented at numbers 0, 5, and 10, respectively. Subjects were asked to identify their level of perceived recovery after warming up and before performing the training protocol.


### 2.2.7. Stroop Test

The Stroop test is a psychological test of mental vitality and flexibility. The test consists of computerized presentation of the names of four colors (yellow, blue, green, and red), written in capital letters, Times New Roman font, size 24, in black. The order is semirandom, so that the same word never appears two consecutive times throughout the test. The subject’s task is to read each word as quickly as possible. This part of the test is intended to obtain a baseline to evaluate the reading ability and determine whether this ability is high enough so as not to hinder the interference effect. This is because the effect of3b2:?thyc=10color-word3b2:?thyc interference may be absent if the reading ability is lower than expected. Characterized by a series of colorized words, which themselves are colors, subjects were instructed to read the color of the word aloud and not the word itself. Correct answers and time to completion were recorded. “Congruent” indicates the number of correct responses out of five responses to the test with matching words and colors. “Incongruent” indicates the number of correct responses out of fifteen responses to the test with nonmatched words and colors.


### 2.2.8. Blood Safety Markers

Subjects donated approximately 10 mL of fasted, whole blood at the baseline and at the end of week 8. All blood samples were collected by a trained phlebotomist via venipuncture of an antecubital vein in the3b2:?A3B2 tlsb=-0.15ptforearm using standard sterile procedures. Biomarkers comprising CMP, CBC, and lipid panels were assayed for data collection.


### 2.2.9. Statistical Analysis

3b2:?A3B2 tlsb=-0.3ptBefore carrying out the parametric statistical analysis, dependent variables were examined for a normal distribution and outliers through investigation of boxplots and a normality test (3b2:?thyc=10i.3b2:?show $132#e.,3b2:?thyc[tnq_nbsp] Shapiro–3b2:?show $132#Wilk). Normality tests revealed no outliers, and analysis was performed using the original data set. Repeated measures ANOVA were used to scrutinize the effects of supplementation on dependent variables assuming the group (placebo and krill oil) and time (pre- and posttest) as fixed factors (GraphPad Prism 7®, La Jolla, CA). Whenever an interaction or main effect was demonstrated, a Bonferroni post hoc test was performed to identify where the differences occurred. The significance level was previously set at p<0.05. All data are reported as mean ± SEM. Analysis of baseline characteristic revealed that there were no statistical significant difference between groups for the investigated dependent variables (p>0.05).


## 2.2.1. Study Design

This study consisted of a randomized,3b2:?thyc=10double-blind3b2:?thyc protocol consisting of 2 groups of individuals 3b2:?A3B2 tlsb=-0.15ptgiven either 3 g of placebo (olive oil) or 3 g of krill (Euphausia superba) oil (Rimfrost Sublime, Rimfrost USA, LLC, Merry Hill, NC, USA, Lot 8723-15-01-03, consisting of 43.8% phospholipids (1.3 g), delivering 963 mg total 3b2:?thyc=10omega-33b2:?thyc fatty acids (240 mg DHA, 393 mg EPA), and 0.54 mg astaxanthin) 3b2:?thyc=10pre-workout3b2:?thyc on training days, and with breakfast on nontraining days, during an 8-week high intensity, nonlinear, periodized resistance training protocol in form of 6 500 mg softgel capsules. The softgel capsules were manufactured by Aenova, Miami, FL, USA. Body composition, maximal strength, peak power, and rate of perceived recovery were assessed collectively at the end of weeks 0 and 8. In addition, safety parameters (comprehensive metabolic panel (CMP), complete blood count (CBC), and urine analysis (UA)) were measured pre- and posttesting.


## 2.2.2. Subjects

Forty subjects were assessed for eligibility and of the3b2:?thyc=10twenty-one3b2:?thyc subjects enrolled 11 subjects received the placebo and 10 received krill oil. A total of 3 subjects were lost to 3b2:?thyc=10follow-up3b2:?thyc by a lack of communication with the researcher (2 from the placebo and 1 from the krill oil group). A total of 18 subjects were analyzed, 9 from each condition (Figure 3b2:?show [?"gr1",5,"cond1"]1).Figure 1
CONSORT chart.3b2:?A3B2 tlsb=-0.29ptSubject inclusion criteria were males 18 to 30 years of age, resistance training at least 2 times per week for the past six months, a minimum of 1 year of training experience active and currently resistance training, free of musculoskeletal, metabolic, and respiratory disorders, free of cardiovascular disease, no musculoskeletal injuries with the last six months, no history of smoking or drug use, no history of excessive alcohol consumption, not taking prescription medication, have not used a fish oil-, thermogenic-, protein-, amino acid-, or creatine supplement within the prior two months, and have not habitually used caffeine (3b2:?thyc=10e.3b2:?show $132#g.,3b2:?thyc[tnq_nbsp] more than 2 cups of coffee per day). Subjects were 3b2:?thyc=10matched-paired3b2:?thyc by age, body mass, strength and resistance training, and physical activity background and then randomly placed into one of the two groups. Subjects were prohibited from consuming any nutritional supplements (including fish oil) for the duration of the study. Additionally, subjects were instructed to avoid consumption of all fish and fish byproducts. After an explanation of the procedures and associated risks, all volunteers completed written informed consent. All procedures were approved by the IntegReview Institutional Review Board, Austin, TX, USA (protocol #7952). This study was registered with the ISRCTN registry (ISRCTN11524409). Subject characteristic data are displayed in Table 3b2:?show [?"tbl1",5,"cond1"]1.Table 1
Subject characteristic data.
Age (yrs)
Training age (yrs)
Body mass (kg)
Height (cm)
3b2:?show "^tbl_width"$01649v
Placebo(n=9)
22.0 ± 0.6
3.0 ± 0.2
79.9 ± 3.9
179.3 ± 3.1
Krill oil(n=9)
22.3 ± 0.4
3.1 ± 0.7
82.9 ± 5.0
178.8 ± 2.3


## 2.2.3. Resistance Exercise Training Protocol

Resistance training occurred four days per week (programmed, nonlinear training split). The resistance training protocol was modified from Kraemer et[tnq_nbsp] al. [22] and Montiero et[tnq_nbsp] al. [23]. These researchers found that a nonlinear resistance training program yielded greater results than a traditional or nonperiodized program in athletes (Montiero et[tnq_nbsp] al. [23]). The program was designed to train all major muscle groups using many compound movements for the upper body (3b2:?thyc=10e.3b2:?show $132#g.,3b2:?thyc[tnq_nbsp] bench press, dips, shoulder press, 3b2:?thyc=10pull-ups,3b2:?thyc and bent over rows), lower body (leg press, leg extensions, and 3b2:?thyc=10glute-ham3b2:?thyc raises), and the core. The programmed, nonlinear training split was divided into hypertrophy days consisting of 8–3b2:?show $132#10 RM loads for 3 sets, with 90 seconds rest, strength endurance days consisting of 12–3b2:?show $132#15 repetitions, with 60 seconds rest, and on days training maximal strength consisting of 3 to 5 RM loads with 3 sets for all exercises except the leg press and bench press (5 total sets). Weights were progressively increased or decreased on a 3b2:?thyc=10set-by-set3b2:?thyc basis by 2–3b2:?show $132#5% to meet prescribed repetitions. Briefly, when subjects could have achieved more[tnq_nbsp] than 2 repetitions over the prescribed number, the load[tnq_nbsp] was 3b2:?A3B2 tlsb .2ptincreased by 2–3b2:?show $132#5%. When subjects failed to reach the prescribed repetitions under their own will, the load was decreased by 2–3b2:?show $132#5%. All training sessions were closely monitored to ensure effort and intensity to maximal on each training sessions. Subjects trained each body 3b2:?A3B2 tlsb=-0.25ptpart twice weekly and alternately between hypertrophy and heavy 3b2:?A3B2 tlsb .25ptworkouts. We have selected this protocol because a 3b2:?thyc=10meta-3b2:?show $132#analysis3b2:?thyc by Rhea [24] indicated that training at this frequency was ideal for moderately resistance trained individuals (Table 2).Table 2
Training Protocol.
Weekly Schedule
Weight Training
Week
1E
2H
3E
4H
Hypertrophy/endurance:
3 sets/12 repetitions60s rest
3 sets/8 repetitions90 s rest
3 sets/15 repetitions60s rest
3 sets/10 repetitions90s rest
Strength:
3–5 sets/5 repetitions3–5 min rest
3–5 sets/4 repetitions3–5 min rest
3–5 sets/3 repetitions3–5 min rest
3–5 sets/5 repetitions3–5 min rest
Week
5
E
6
H
7
E
8
H
Hypertrophy/endurance:
3 sets/12 repetitions90 s rest
3 sets/8 repetitions90s rest
3 sets/15 repetitions60s rest
3 sets/10 repetitions90s rest
Strength:
3–5 sets/ 4 repetitions3–5 min rest
3–5 sets/4 repetitions3–5 min rest
3–5 sets/ 3 repetitions3–5 min rest
3–5 sets/ 3 repetitions3–5 min rest
Exercise Selection
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
Strength
Rest
Hypertrophy/endurance
Hypertrophy/endurance
Rest
Strength
Leg press
Bench press
Squat
Bench press
Squat
BB row
Deadlift
BB row
3b2:?A3B2 tlsb -0.25ptGlute-ham raise
BB shoulder press
Leg press
BB shoulder press
Pull-ups
Glute-ham raise
Pull-ups
DB press
Seated leg curl
Incline DB press
Lying leg curl
Side lateral raise
Leg ext.
Preacher curl
Calf raise
Overhead tricep ext.
Cable fly
Hammer curl
Tricep pushdown
Cable press


## 2.2.4. Body Composition

3b2:?A3B2 tlsb 0.1ptA 3b2:?thyc=10whole-body3b2:?thyc 3b2:?thyc=10dual-energy3b2:?thyc X-3b2:?show $132#ray absorptiometry (DXA) (Hologic, Bedford, MA, USA) scan was utilized to measure body composition. Lean body mass (LBM) and fat mass (FM) were determined for the total body with the subject laying in a supine position with the knee extended and instructed not to move for the entire duration of the scan (∼5 minutes). Results from each scan were uploaded and accessed on a computer directly connected to the DXA device. All DXA scans were conducted prior to and after the completion of the study, and each subject was required to fast overnight (10 hours) prior to the DXA scan. Calibration of the DXA device was done against a phantom provided by the manufacturing company prior to testing.


## 2.2.5.3b2:?A3B2 tlsb -.21ptStrength Assessment

3b2:?thyc=10One-repetition3b2:?thyc maximum (1 RM) 3b2:?show [?tjl=20mm]&tjlpc;[?tjl]was assessed on bench press and leg press at baseline and after 8 weeks. Loads were increased incrementally until maximal load or failure at a given load was reached. Briefly, subjects performed a general warmup and a specific warmup consisting of three sets. During the first set, subjects performed 10 repetitions with 50% of their predicted 1 RM. For the second set, they performed five repetitions with 70% of the predicted 1 RM. In the third set, subjects perform one repetition with 90% of their predicted 1 RM. After the completion of warmup sets, subjects rested for 3 minutes. Then, each subject had as many as five attempts to achieve their 1 RM load with 3–3b2:?show $132#5 minutes rest between each attempt.


## 2.2.6. Perceptual Measures

The perceptual measures were collected using a perceived recovery status scale. Ratings of perceived recovery were collected at the beginning and end of[tnq_nbsp] every week. The perceived recovery status scale consisted of a scalar representation numbering from 0 to 10. Visual descriptors of “very poorly recovered,” “adequately recovered,” and “very well recovered” for perceived recovery were presented at numbers 0, 5, and 10, respectively. Subjects were asked to identify their level of perceived recovery after warming up and before performing the training protocol.


## 2.2.7. Stroop Test

The Stroop test is a psychological test of mental vitality and flexibility. The test consists of computerized presentation of the names of four colors (yellow, blue, green, and red), written in capital letters, Times New Roman font, size 24, in black. The order is semirandom, so that the same word never appears two consecutive times throughout the test. The subject’s task is to read each word as quickly as possible. This part of the test is intended to obtain a baseline to evaluate the reading ability and determine whether this ability is high enough so as not to hinder the interference effect. This is because the effect of3b2:?thyc=10color-word3b2:?thyc interference may be absent if the reading ability is lower than expected. Characterized by a series of colorized words, which themselves are colors, subjects were instructed to read the color of the word aloud and not the word itself. Correct answers and time to completion were recorded. “Congruent” indicates the number of correct responses out of five responses to the test with matching words and colors. “Incongruent” indicates the number of correct responses out of fifteen responses to the test with nonmatched words and colors.


## 2.2.8. Blood Safety Markers

Subjects donated approximately 10 mL of fasted, whole blood at the baseline and at the end of week 8. All blood samples were collected by a trained phlebotomist via venipuncture of an antecubital vein in the3b2:?A3B2 tlsb=-0.15ptforearm using standard sterile procedures. Biomarkers comprising CMP, CBC, and lipid panels were assayed for data collection.


## 2.2.9. Statistical Analysis

3b2:?A3B2 tlsb=-0.3ptBefore carrying out the parametric statistical analysis, dependent variables were examined for a normal distribution and outliers through investigation of boxplots and a normality test (3b2:?thyc=10i.3b2:?show $132#e.,3b2:?thyc[tnq_nbsp] Shapiro–3b2:?show $132#Wilk). Normality tests revealed no outliers, and analysis was performed using the original data set. Repeated measures ANOVA were used to scrutinize the effects of supplementation on dependent variables assuming the group (placebo and krill oil) and time (pre- and posttest) as fixed factors (GraphPad Prism 7®, La Jolla, CA). Whenever an interaction or main effect was demonstrated, a Bonferroni post hoc test was performed to identify where the differences occurred. The significance level was previously set at p<0.05. All data are reported as mean ± SEM. Analysis of baseline characteristic revealed that there were no statistical significant difference between groups for the investigated dependent variables (p>0.05).


## 3. Results

### 3.1. Cell Culture Tests

As shown in Figure3b2:?show [?"gr2",5,"cond1"]2, S-3b2:?show $132#PC showed no increase in the ratio of P-3b2:?show $132#3b2:?thyc=10p70-3893b2:?thyc to total p70 compared to vehicle (control) stimulated cells. In contrast, elevated mTOR signaling was observed at all tested concentrations of krill 3b2:?thyc=10oil-PC3b2:?thyc (+187% and +242% resp.; p<0.001).Figure 2
3b2:?A3B2 tlsb=-0.15ptThe effect of phosphatidylcholine on the activation of mTOR signaling. C2C12 myoblasts were stimulated for 20 minutes with vehicle (control), or 10–3b2:?show $132#30 µM of S-PC, or krill oil-derived PC. The samples were then subjected to the western blot analysis for p70 phosphorylated on the threonine 389 residue (P-p70(389)) and total p70. The ratio of these signals was calculated and used as a marker of mTOR signaling. Values in the graphs represent the mean + SEM and were obtained from 2 to 3 independent experiments (n = 3–3b2:?show $132#6/group). ∗Significantly different from the control and S-PC (p<0.05).


### 3.2. Human Efficacy Study

#### 3.2.1. Body Composition

3b2:?A3B2 tlsb=-0.25ptA significant main time effect was noted for lean mass (p=0.021); however, there were no differences between the groups (placebo: meandiff 0.3 kg, +0.5%; 3b2:?A3B2 tlsb .1ptkrill oil: meandiff 1.4 kg, +2.1%, Figure 3b2:?show [?"gr3",5,"cond1"]3). Regarding the main time effect for lean mass, post hoc analysis indicated a 3b2:?thyc=10within-group3b2:?thyc difference from 3b2:?thyc=10pre-to3b2:?thyc posttesting for krill oil (p=0.024, Figure 3(a)).3b2:?tex type="figure" layout="twocols" n="1,2"Figure 3
Summary of the group lean mass values (a), individual lean mass response (b), and lean mass delta change (c) (∗significant within-group difference (p<0.05)).
(a)
(b)
(c)No between or within group differences were observed for fat mass (p>0.05, placebo: meandiff 0.3 kg, +0.3%; krill oil: meandiff −0.6 kg, −3.6%, Figure 3b2:?show [?"gr4",5,"cond1"]4).3b2:?tex type="figure" layout="twocols" n="1,2"Figure 4
3b2:?A3B2 tlsb=0.15ptSummary of group fat mass values (a), individual fat mass response (b), and fat mass delta change (c).
(a)
(b)
(c)


#### 3.2.2. Muscle Strength

A significant main time effect was demonstrated whereby both conditions increased performance on bench press (p<0.0001, placebo: meandiff 3.4 kg, +3.5%; group krill oil: meandiff 4.2 kg, +4.3%, Figure 3b2:?show [?"gr5",5,"cond1"]5) and leg press (p<0.0001, placebo: meandiff 44.3 kg, +15.8%; krill[tnq_nbsp] oil: meandiff 49.0 kg, +15.8%, Figure 3b2:?show [?"gr6",5,"cond1"]6) from 3b2:?thyc=10pre-to3b2:?thyc posttesting.3b2:?tex type="figure" layout="twocols" n="1,2"Figure 5
Summary of group bench-press performance (a), individual bench-press performance (b), and bench-press performance delta change (c) (∗significant time effect whereby each group increased from baseline (p<0.001)).
(a)
(b)
(c)3b2:?tex type="figure" layout="twocols" n="1,2"Figure 6
Summary of group leg-press performance (a) and individual leg-press performance (b), and leg-press performance delta change (c) (∗significant time effect whereby each group increased from baseline (p<0.001)).
(a)
(b)
(c)


#### 3.2.3. Perceptual Measures

3b2:?A3B2 tlsb=0.15ptA main time effect was demonstrated for perceived recovery (p<0.001). Placebo had a significantly higher perceived recovery at week 8 compared to baseline and weeks 3, 4, and 6; while weeks 2, 5, 3b2:?A3B2 tlsb=-0.15ptand 7 were also higher than week 6. Krill oil had a significantly 3b2:?A3B2 tlsb=0.15pthigher perceived recovery at week 8 compared to baseline and weeks 4, 5, and 6; while weeks 1, 2, and 3 were also higher than week 6 (Figure 3b2:?show [?"gr7",5,"cond1"]7). There were no between group differences for perceived recovery at any time point (Figure 7).Figure 7
Summary plot for perceived recovery scale weekly average (∗significantly higher than baseline, a = significantly lower than week 8 (p<0.05), and b = significantly higher than week 6 (p<0.05)).


#### 3.2.4. Stroop Test

A main time effect was demonstrated for the Stroop test completion time (p=0.007), and post hoc analysis indicated that each group demonstrated strong trends for 3b2:?thyc=10within-group3b2:?thyc differences from 3b2:?thyc=10pre- to3b2:?thyc posttesting (placebo, p=0.087; krill oil, p=0.085) (Table 3b2:?show [?"tbl2",5,"cond1"]3).Table 3
Stroop test results.
Placebo
Krill oil
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
Congruent
5.0 ± 0.0
5.0 ± 0.0
5.0 ± 0.0
5.0 ± 0.0
Incongruent
14.7 ± 0.2
14.8 ± 0.2
14.8 ± 0.2
14.8 ± 0.2
Completion time
32.6 ± 6.7
17.2 ± 2.3 ^
33.8 ± 6.1
18.3 ± 2.1 ^
^3b2:?A3B2 h 2ptTrend for within-group significant differences from pretest (p<0.1).


#### 3.2.5. Blood Safety Markers

There were no within or between group differences for markers on the comprehensive metabolic panel (Table3b2:?show [?"tbl3",5,"cond1"]4), complete blood count (Table 3b2:?show [?"tbl4",5,"cond1"]5), or urine analysis (not depicted) (p>0.05).Table 4
Comprehensive metabolic panel.
Placebo
Krill oil
3b2:?A3B2 tbcfmtv c
G × T p value
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
Glucose (mg/dL)
84 ± 2
86 ± 4
91 ± 3
90 ± 4
0.689
BUN (mg/dL)
13 ± 1
13 ± 1
13 ± 2
15 ± 2
0.200
Creatinine (mg/dL)
1.07 ± 0.1
1.05 ± 0.1
1.06 ± 0.1
1.00 ± 0.1
0.523
Total protein (g/dL)
7.1 ± 0.1
7.1 ± 0.1
7.2 ± 0.1
7.0 ± 0.1
0.114
Albumin (g/dL)
4.6 ± 0.1
4.5 ± 0.1
4.8 ± 0.1
4.6 ± 0.1
0.227
Total bilirubin (mg/dL)
0.4 ± 0.1
0.3 ± 0.1
0.6 ± 0.3
0.5 ± 0.3
0.510
Sodium (mmol/L)
141 ± 1
141 ± 1
142 ± 1
141 ± 1
0.339
Potassium (mmol/L)
4.3 ± 0.1
4.2 ± 0.1
4.3 ± 0.1
4.3 ± 0.1
0.328
Chloride (mmol/L)
100 ± 1
101 ± 1
101 ± 1
101 ± 1
0.939
CO2 (mmol/L)
23 ± 1
22 ± 1
22 ± 1
22 ± 1
0.791
ALP (IU/L)
73 ± 6
75 ± 6
75 ± 5
70 ± 6
0.075
AST (IU/L)
24 ± 5
22 ± 2
20 ± 1
17 ± 1
0.827
ALT (IU/L)
22 ± 5
19 ± 3
19 ± 1
17 ± 2
0.754
Values are reported as mean ± standard deviation; BUN, blood urea nitrogen; CO2, carbon dioxide; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine transaminase.Table 5
Complete blood count panel
Placebo
Krill oil
G × T p value
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
WBC (K/µL)
5.6 ± 0.2
5.5 ± 0.2
5.7 ± 0.2
5.5 ± 0.3
0.586
RBC (M/µL)
5.01 ± 0.14
5.12 ± 0.12
5.26 ± 0.08
5.08 ± 0.09
0.114
Hemoglobin (g/dL)
15.1 ± 0.4
15.4 ± 0.2
14.7 ± 1.1
15.3 ± 0.3
0.785
Hematocrit (%)
44.6 ± 1.2
45.3 ± 0.7
46.9 ± 0.9
45.7 ± 1.1
0.160
MVC (fL)
89.1 ± 1.6
89.2 ± 1.1
88.8 ± 1.3
90.4 ± 1.2
0.414
MCH (pg)
30.2 ± 0.7
29.9 ± 0.5
30.0 ± 0.6
30.0 ± 0.4
0.999
MCHC (g/dL)
33.4 ± 0.2
33.2 ± 0.3
33.8 ± 0.3
33.3 ± 0.2
0.481
Platelets (K/µL)
228 ± 10
221 ± 10
232 ± 11
219 ± 10
0.431
Neutrophils (K/µL)
2.8 ± 0.2
2.9 ± 0.2
3.1 ± 0.2
2.8 ± 0.2
0.197
Lymphocytes (K/µL)
2.1 ± 0.1
2.0 ± 0.1
2.0 ± 0.1
2.0 ± 0.1
0.999
Monocytes (K/µL)
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.2
0.739
Eosinophils (K/µL)
0.1 ± 0.1
0.2 ± 0.1
0.1 ± 0.1
0.1 ± 0.1
0.461
Basophils (K/µL)
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.555
Values are reported as mean ± standard deviation; WBC, white blood cells; RBC,  red blood cells; MVC, mean cell volume; MCH, mean cell hemoglobin; MCHC,  mean cell hemoglobin concentration.


## 3.1. Cell Culture Tests

As shown in Figure3b2:?show [?"gr2",5,"cond1"]2, S-3b2:?show $132#PC showed no increase in the ratio of P-3b2:?show $132#3b2:?thyc=10p70-3893b2:?thyc to total p70 compared to vehicle (control) stimulated cells. In contrast, elevated mTOR signaling was observed at all tested concentrations of krill 3b2:?thyc=10oil-PC3b2:?thyc (+187% and +242% resp.; p<0.001).Figure 2
3b2:?A3B2 tlsb=-0.15ptThe effect of phosphatidylcholine on the activation of mTOR signaling. C2C12 myoblasts were stimulated for 20 minutes with vehicle (control), or 10–3b2:?show $132#30 µM of S-PC, or krill oil-derived PC. The samples were then subjected to the western blot analysis for p70 phosphorylated on the threonine 389 residue (P-p70(389)) and total p70. The ratio of these signals was calculated and used as a marker of mTOR signaling. Values in the graphs represent the mean + SEM and were obtained from 2 to 3 independent experiments (n = 3–3b2:?show $132#6/group). ∗Significantly different from the control and S-PC (p<0.05).


## 3.2. Human Efficacy Study

### 3.2.1. Body Composition

3b2:?A3B2 tlsb=-0.25ptA significant main time effect was noted for lean mass (p=0.021); however, there were no differences between the groups (placebo: meandiff 0.3 kg, +0.5%; 3b2:?A3B2 tlsb .1ptkrill oil: meandiff 1.4 kg, +2.1%, Figure 3b2:?show [?"gr3",5,"cond1"]3). Regarding the main time effect for lean mass, post hoc analysis indicated a 3b2:?thyc=10within-group3b2:?thyc difference from 3b2:?thyc=10pre-to3b2:?thyc posttesting for krill oil (p=0.024, Figure 3(a)).3b2:?tex type="figure" layout="twocols" n="1,2"Figure 3
Summary of the group lean mass values (a), individual lean mass response (b), and lean mass delta change (c) (∗significant within-group difference (p<0.05)).
(a)
(b)
(c)No between or within group differences were observed for fat mass (p>0.05, placebo: meandiff 0.3 kg, +0.3%; krill oil: meandiff −0.6 kg, −3.6%, Figure 3b2:?show [?"gr4",5,"cond1"]4).3b2:?tex type="figure" layout="twocols" n="1,2"Figure 4
3b2:?A3B2 tlsb=0.15ptSummary of group fat mass values (a), individual fat mass response (b), and fat mass delta change (c).
(a)
(b)
(c)


### 3.2.2. Muscle Strength

A significant main time effect was demonstrated whereby both conditions increased performance on bench press (p<0.0001, placebo: meandiff 3.4 kg, +3.5%; group krill oil: meandiff 4.2 kg, +4.3%, Figure 3b2:?show [?"gr5",5,"cond1"]5) and leg press (p<0.0001, placebo: meandiff 44.3 kg, +15.8%; krill[tnq_nbsp] oil: meandiff 49.0 kg, +15.8%, Figure 3b2:?show [?"gr6",5,"cond1"]6) from 3b2:?thyc=10pre-to3b2:?thyc posttesting.3b2:?tex type="figure" layout="twocols" n="1,2"Figure 5
Summary of group bench-press performance (a), individual bench-press performance (b), and bench-press performance delta change (c) (∗significant time effect whereby each group increased from baseline (p<0.001)).
(a)
(b)
(c)3b2:?tex type="figure" layout="twocols" n="1,2"Figure 6
Summary of group leg-press performance (a) and individual leg-press performance (b), and leg-press performance delta change (c) (∗significant time effect whereby each group increased from baseline (p<0.001)).
(a)
(b)
(c)


### 3.2.3. Perceptual Measures

3b2:?A3B2 tlsb=0.15ptA main time effect was demonstrated for perceived recovery (p<0.001). Placebo had a significantly higher perceived recovery at week 8 compared to baseline and weeks 3, 4, and 6; while weeks 2, 5, 3b2:?A3B2 tlsb=-0.15ptand 7 were also higher than week 6. Krill oil had a significantly 3b2:?A3B2 tlsb=0.15pthigher perceived recovery at week 8 compared to baseline and weeks 4, 5, and 6; while weeks 1, 2, and 3 were also higher than week 6 (Figure 3b2:?show [?"gr7",5,"cond1"]7). There were no between group differences for perceived recovery at any time point (Figure 7).Figure 7
Summary plot for perceived recovery scale weekly average (∗significantly higher than baseline, a = significantly lower than week 8 (p<0.05), and b = significantly higher than week 6 (p<0.05)).


### 3.2.4. Stroop Test

A main time effect was demonstrated for the Stroop test completion time (p=0.007), and post hoc analysis indicated that each group demonstrated strong trends for 3b2:?thyc=10within-group3b2:?thyc differences from 3b2:?thyc=10pre- to3b2:?thyc posttesting (placebo, p=0.087; krill oil, p=0.085) (Table 3b2:?show [?"tbl2",5,"cond1"]3).Table 3
Stroop test results.
Placebo
Krill oil
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
Congruent
5.0 ± 0.0
5.0 ± 0.0
5.0 ± 0.0
5.0 ± 0.0
Incongruent
14.7 ± 0.2
14.8 ± 0.2
14.8 ± 0.2
14.8 ± 0.2
Completion time
32.6 ± 6.7
17.2 ± 2.3 ^
33.8 ± 6.1
18.3 ± 2.1 ^
^3b2:?A3B2 h 2ptTrend for within-group significant differences from pretest (p<0.1).


### 3.2.5. Blood Safety Markers

There were no within or between group differences for markers on the comprehensive metabolic panel (Table3b2:?show [?"tbl3",5,"cond1"]4), complete blood count (Table 3b2:?show [?"tbl4",5,"cond1"]5), or urine analysis (not depicted) (p>0.05).Table 4
Comprehensive metabolic panel.
Placebo
Krill oil
3b2:?A3B2 tbcfmtv c
G × T p value
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
Glucose (mg/dL)
84 ± 2
86 ± 4
91 ± 3
90 ± 4
0.689
BUN (mg/dL)
13 ± 1
13 ± 1
13 ± 2
15 ± 2
0.200
Creatinine (mg/dL)
1.07 ± 0.1
1.05 ± 0.1
1.06 ± 0.1
1.00 ± 0.1
0.523
Total protein (g/dL)
7.1 ± 0.1
7.1 ± 0.1
7.2 ± 0.1
7.0 ± 0.1
0.114
Albumin (g/dL)
4.6 ± 0.1
4.5 ± 0.1
4.8 ± 0.1
4.6 ± 0.1
0.227
Total bilirubin (mg/dL)
0.4 ± 0.1
0.3 ± 0.1
0.6 ± 0.3
0.5 ± 0.3
0.510
Sodium (mmol/L)
141 ± 1
141 ± 1
142 ± 1
141 ± 1
0.339
Potassium (mmol/L)
4.3 ± 0.1
4.2 ± 0.1
4.3 ± 0.1
4.3 ± 0.1
0.328
Chloride (mmol/L)
100 ± 1
101 ± 1
101 ± 1
101 ± 1
0.939
CO2 (mmol/L)
23 ± 1
22 ± 1
22 ± 1
22 ± 1
0.791
ALP (IU/L)
73 ± 6
75 ± 6
75 ± 5
70 ± 6
0.075
AST (IU/L)
24 ± 5
22 ± 2
20 ± 1
17 ± 1
0.827
ALT (IU/L)
22 ± 5
19 ± 3
19 ± 1
17 ± 2
0.754
Values are reported as mean ± standard deviation; BUN, blood urea nitrogen; CO2, carbon dioxide; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine transaminase.Table 5
Complete blood count panel
Placebo
Krill oil
G × T p value
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
WBC (K/µL)
5.6 ± 0.2
5.5 ± 0.2
5.7 ± 0.2
5.5 ± 0.3
0.586
RBC (M/µL)
5.01 ± 0.14
5.12 ± 0.12
5.26 ± 0.08
5.08 ± 0.09
0.114
Hemoglobin (g/dL)
15.1 ± 0.4
15.4 ± 0.2
14.7 ± 1.1
15.3 ± 0.3
0.785
Hematocrit (%)
44.6 ± 1.2
45.3 ± 0.7
46.9 ± 0.9
45.7 ± 1.1
0.160
MVC (fL)
89.1 ± 1.6
89.2 ± 1.1
88.8 ± 1.3
90.4 ± 1.2
0.414
MCH (pg)
30.2 ± 0.7
29.9 ± 0.5
30.0 ± 0.6
30.0 ± 0.4
0.999
MCHC (g/dL)
33.4 ± 0.2
33.2 ± 0.3
33.8 ± 0.3
33.3 ± 0.2
0.481
Platelets (K/µL)
228 ± 10
221 ± 10
232 ± 11
219 ± 10
0.431
Neutrophils (K/µL)
2.8 ± 0.2
2.9 ± 0.2
3.1 ± 0.2
2.8 ± 0.2
0.197
Lymphocytes (K/µL)
2.1 ± 0.1
2.0 ± 0.1
2.0 ± 0.1
2.0 ± 0.1
0.999
Monocytes (K/µL)
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.2
0.739
Eosinophils (K/µL)
0.1 ± 0.1
0.2 ± 0.1
0.1 ± 0.1
0.1 ± 0.1
0.461
Basophils (K/µL)
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.555
Values are reported as mean ± standard deviation; WBC, white blood cells; RBC,  red blood cells; MVC, mean cell volume; MCH, mean cell hemoglobin; MCHC,  mean cell hemoglobin concentration.


## 3.2.1. Body Composition

3b2:?A3B2 tlsb=-0.25ptA significant main time effect was noted for lean mass (p=0.021); however, there were no differences between the groups (placebo: meandiff 0.3 kg, +0.5%; 3b2:?A3B2 tlsb .1ptkrill oil: meandiff 1.4 kg, +2.1%, Figure 3b2:?show [?"gr3",5,"cond1"]3). Regarding the main time effect for lean mass, post hoc analysis indicated a 3b2:?thyc=10within-group3b2:?thyc difference from 3b2:?thyc=10pre-to3b2:?thyc posttesting for krill oil (p=0.024, Figure 3(a)).3b2:?tex type="figure" layout="twocols" n="1,2"Figure 3
Summary of the group lean mass values (a), individual lean mass response (b), and lean mass delta change (c) (∗significant within-group difference (p<0.05)).
(a)
(b)
(c)No between or within group differences were observed for fat mass (p>0.05, placebo: meandiff 0.3 kg, +0.3%; krill oil: meandiff −0.6 kg, −3.6%, Figure 3b2:?show [?"gr4",5,"cond1"]4).3b2:?tex type="figure" layout="twocols" n="1,2"Figure 4
3b2:?A3B2 tlsb=0.15ptSummary of group fat mass values (a), individual fat mass response (b), and fat mass delta change (c).
(a)
(b)
(c)


## 3.2.2. Muscle Strength

A significant main time effect was demonstrated whereby both conditions increased performance on bench press (p<0.0001, placebo: meandiff 3.4 kg, +3.5%; group krill oil: meandiff 4.2 kg, +4.3%, Figure 3b2:?show [?"gr5",5,"cond1"]5) and leg press (p<0.0001, placebo: meandiff 44.3 kg, +15.8%; krill[tnq_nbsp] oil: meandiff 49.0 kg, +15.8%, Figure 3b2:?show [?"gr6",5,"cond1"]6) from 3b2:?thyc=10pre-to3b2:?thyc posttesting.3b2:?tex type="figure" layout="twocols" n="1,2"Figure 5
Summary of group bench-press performance (a), individual bench-press performance (b), and bench-press performance delta change (c) (∗significant time effect whereby each group increased from baseline (p<0.001)).
(a)
(b)
(c)3b2:?tex type="figure" layout="twocols" n="1,2"Figure 6
Summary of group leg-press performance (a) and individual leg-press performance (b), and leg-press performance delta change (c) (∗significant time effect whereby each group increased from baseline (p<0.001)).
(a)
(b)
(c)


## 3.2.3. Perceptual Measures

3b2:?A3B2 tlsb=0.15ptA main time effect was demonstrated for perceived recovery (p<0.001). Placebo had a significantly higher perceived recovery at week 8 compared to baseline and weeks 3, 4, and 6; while weeks 2, 5, 3b2:?A3B2 tlsb=-0.15ptand 7 were also higher than week 6. Krill oil had a significantly 3b2:?A3B2 tlsb=0.15pthigher perceived recovery at week 8 compared to baseline and weeks 4, 5, and 6; while weeks 1, 2, and 3 were also higher than week 6 (Figure 3b2:?show [?"gr7",5,"cond1"]7). There were no between group differences for perceived recovery at any time point (Figure 7).Figure 7
Summary plot for perceived recovery scale weekly average (∗significantly higher than baseline, a = significantly lower than week 8 (p<0.05), and b = significantly higher than week 6 (p<0.05)).


## 3.2.4. Stroop Test

A main time effect was demonstrated for the Stroop test completion time (p=0.007), and post hoc analysis indicated that each group demonstrated strong trends for 3b2:?thyc=10within-group3b2:?thyc differences from 3b2:?thyc=10pre- to3b2:?thyc posttesting (placebo, p=0.087; krill oil, p=0.085) (Table 3b2:?show [?"tbl2",5,"cond1"]3).Table 3
Stroop test results.
Placebo
Krill oil
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
Congruent
5.0 ± 0.0
5.0 ± 0.0
5.0 ± 0.0
5.0 ± 0.0
Incongruent
14.7 ± 0.2
14.8 ± 0.2
14.8 ± 0.2
14.8 ± 0.2
Completion time
32.6 ± 6.7
17.2 ± 2.3 ^
33.8 ± 6.1
18.3 ± 2.1 ^
^3b2:?A3B2 h 2ptTrend for within-group significant differences from pretest (p<0.1).


## 3.2.5. Blood Safety Markers

There were no within or between group differences for markers on the comprehensive metabolic panel (Table3b2:?show [?"tbl3",5,"cond1"]4), complete blood count (Table 3b2:?show [?"tbl4",5,"cond1"]5), or urine analysis (not depicted) (p>0.05).Table 4
Comprehensive metabolic panel.
Placebo
Krill oil
3b2:?A3B2 tbcfmtv c
G × T p value
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
Glucose (mg/dL)
84 ± 2
86 ± 4
91 ± 3
90 ± 4
0.689
BUN (mg/dL)
13 ± 1
13 ± 1
13 ± 2
15 ± 2
0.200
Creatinine (mg/dL)
1.07 ± 0.1
1.05 ± 0.1
1.06 ± 0.1
1.00 ± 0.1
0.523
Total protein (g/dL)
7.1 ± 0.1
7.1 ± 0.1
7.2 ± 0.1
7.0 ± 0.1
0.114
Albumin (g/dL)
4.6 ± 0.1
4.5 ± 0.1
4.8 ± 0.1
4.6 ± 0.1
0.227
Total bilirubin (mg/dL)
0.4 ± 0.1
0.3 ± 0.1
0.6 ± 0.3
0.5 ± 0.3
0.510
Sodium (mmol/L)
141 ± 1
141 ± 1
142 ± 1
141 ± 1
0.339
Potassium (mmol/L)
4.3 ± 0.1
4.2 ± 0.1
4.3 ± 0.1
4.3 ± 0.1
0.328
Chloride (mmol/L)
100 ± 1
101 ± 1
101 ± 1
101 ± 1
0.939
CO2 (mmol/L)
23 ± 1
22 ± 1
22 ± 1
22 ± 1
0.791
ALP (IU/L)
73 ± 6
75 ± 6
75 ± 5
70 ± 6
0.075
AST (IU/L)
24 ± 5
22 ± 2
20 ± 1
17 ± 1
0.827
ALT (IU/L)
22 ± 5
19 ± 3
19 ± 1
17 ± 2
0.754
Values are reported as mean ± standard deviation; BUN, blood urea nitrogen; CO2, carbon dioxide; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine transaminase.Table 5
Complete blood count panel
Placebo
Krill oil
G × T p value
Pre
Post
Pre
Post
3b2:?show "^tbl_width"$01649v
WBC (K/µL)
5.6 ± 0.2
5.5 ± 0.2
5.7 ± 0.2
5.5 ± 0.3
0.586
RBC (M/µL)
5.01 ± 0.14
5.12 ± 0.12
5.26 ± 0.08
5.08 ± 0.09
0.114
Hemoglobin (g/dL)
15.1 ± 0.4
15.4 ± 0.2
14.7 ± 1.1
15.3 ± 0.3
0.785
Hematocrit (%)
44.6 ± 1.2
45.3 ± 0.7
46.9 ± 0.9
45.7 ± 1.1
0.160
MVC (fL)
89.1 ± 1.6
89.2 ± 1.1
88.8 ± 1.3
90.4 ± 1.2
0.414
MCH (pg)
30.2 ± 0.7
29.9 ± 0.5
30.0 ± 0.6
30.0 ± 0.4
0.999
MCHC (g/dL)
33.4 ± 0.2
33.2 ± 0.3
33.8 ± 0.3
33.3 ± 0.2
0.481
Platelets (K/µL)
228 ± 10
221 ± 10
232 ± 11
219 ± 10
0.431
Neutrophils (K/µL)
2.8 ± 0.2
2.9 ± 0.2
3.1 ± 0.2
2.8 ± 0.2
0.197
Lymphocytes (K/µL)
2.1 ± 0.1
2.0 ± 0.1
2.0 ± 0.1
2.0 ± 0.1
0.999
Monocytes (K/µL)
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.1
0.5 ± 0.2
0.739
Eosinophils (K/µL)
0.1 ± 0.1
0.2 ± 0.1
0.1 ± 0.1
0.1 ± 0.1
0.461
Basophils (K/µL)
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.555
Values are reported as mean ± standard deviation; WBC, white blood cells; RBC,  red blood cells; MVC, mean cell volume; MCH, mean cell hemoglobin; MCHC,  mean cell hemoglobin concentration.


## 4. Discussion

### 4.1. mTOR Activation

Previous studies have shown that3b2:?thyc=10soy-derived3b2:?thyc phosphatidic acid (PA), 3b2:?thyc=10lyso-PA,3b2:?thyc or phosphatidylserine (PS) can stimulate a robust increase in mTOR signaling; however, 3b2:?thyc=10soy-derived3b2:?thyc PC was not effective [21]. While 3b2:?thyc=10egg-derived3b2:?thyc PA increases mTOR signaling, its effects are inferior to 3b2:?thyc=10soy-derived3b2:?thyc PA. 3b2:?thyc=10Egg-derived3b2:?thyc PA has a different fatty acid composition in comparison to 3b2:?thyc=10soy-derived3b2:?thyc PA, containing 3b2:?thyc=10omega-33b2:?thyc fatty acids, such as DHA or EPA, indicating that the fatty acid composition of the phospholipid influences mTOR signaling and 3b2:?thyc=10omega-3s3b2:?thyc likely attenuates the stimulatory effect. Krill oil is a complex mixture of different ingredients with PC being the main phospholipid. Krill 3b2:?thyc=10oil-PC,3b2:?thyc like 3b2:?thyc=10egg-derived3b2:?thyc phospholipids, is rich in 3b2:?thyc=10omega-33b2:?thyc fatty acids which suggest that other ingredients in krill oil such as PA, PS, or even astaxanthin or the combination of these ingredients, might be responsible for the effect on mTOR signaling, as astaxanthin has recently been shown to increase mTOR expressions in mice [25].3b2:?A3B2 tpb -6pt


### 4.2.3b2:?A3B2 tlsb=-0.36ptBody Composition and Athletic Performance

Phospholipids, including PC, PS, or PA, have previously been shown to improve athletic performance [21,[tnq_nbsp] 26]. PS has been shown to reduce 3b2:?thyc=10exercise-induced3b2:?thyc increases in the catabolic hormone cortisol [27] and reduce muscle soreness [28]. PA enhances 3b2:?thyc=10exercise-stimulated3b2:?thyc increases in lean body mass through activation of the mTOR pathway [21], and PC has been shown to maintain choline concentration during exercise [29]. Muscle contractions are induced by signals carried along cholinergic nerves to the muscle fiber, and strenuous exercise has been reported to result in decreased choline concentrations, which negatively influence the rates in which the neurotransmitter acetylcholine is synthesized and released. Maintaining choline levels during exercise has been linked to an increase in athletic performance. 3 g of krill oil provides 1.3 g of phospholipids. The phospholipids in krill oil consist of 90.5% choline containing phospholipids (76.1% PC, 7.0% alkyl acyl PC, 6.5% 3b2:?thyc=10lyso-PC,3b2:?thyc and 0.9% 3b2:?thyc=10lyso-alkyl3b2:?thyc acyl PC), 8.3% ethanolamine containing phospholipids (3.2% phosphatidylethanolamine (PE), 2.3% cardiolipin n-3b2:?show $132#acetyl PE 3b2:?thyc=10(CL/NAPE), 3b2:?thyc1.9% alkyl acyl PE, 0.7% 3b2:?thyc=10lyso-PE,3b2:?thyc and 0.2% 3b2:?thyc=10lyso-alkyl3b2:?thyc acyl PE), 0.7% phosphatidylinositol (PI), and 0.6% PS [30]. The 1.2 g phosphatidylcholine from 3 g of krill oil provides 160 mg choline, and krill oil contains 7.8 mg 3b2:?A3B2 tlsb -.1ptPS and 0.54 mg of astaxanthin. The concentration of all individual active ingredients in 3 g krill oil is lower than the currently established individual effective dose (600 mg of PS to lower cortisol levels, 4 mg of astaxanthin, or 0.2 g per kg body weight of PC). A potential synergy of the different active ingredients at low doses could not be established in this trial.While lean body mass significantly increased in the krill oil group (+1.4 kg, +2.1%) we observed no statistically significant difference in comparison to the control group (+0.3 kg, +0.5%). The increase in lean body mass was matched by an increase in muscle strength in the krill oil group (bench press +4.3 kg (+4.4%); leg press: +48.9 kg (+15.8%)); however, the increase was no different from the control group (+3.4 kg (+3.3%); leg press: +44.2 kg (+14.6%)). Perceived recovery significantly increased in the krill oil group (+15%) at the end of the trial in comparison to baseline; however, the results were not statistically different from the control group (+14%). Perceived recovery was the lowest in both groups in week 6, and subjects seem to adapt to recovering to the training stimulus by the end of the study. While the training protocol was challenging enough to see significant increases in strength, the training stimulus or the recovery periods might not have been challenging enough to elicit differences in perceived recovery. Our results contrast with previous studies showing significant improvements of postexercise muscle soreness with3b2:?thyc=10omega-33b2:?thyc supplementation [4–3b2:?show $132#8], which might be due to the lower dose of 3b2:?thyc=10omega-3s3b2:?thyc administered in our study, 963 mg for 8 weeks versus 3b2:?A3B2 tlsb -.1pt2.4–3b2:?show $132#6 3b2:?thyc=10g/d 3b2:?thycfor 1 to 8 weeks, or the difference in training stimulus and recovery periods. Based on a post hoc power analysis the sample size needed to reach statistical significance would be between 34 and 84 subjects for changes in lean body mass, depending on the level of statistical power (3b2:?thyc=10i.3b2:?show $132#e.,3b2:?thyc[tnq_nbsp] 50% to 90% probability of detecting a significant difference at an alpha level of 0.05). Future studies should investigate the activation of mTOR in vivo through a muscular biopsy.3b2:?A3B2 tpb 1pt


### 4.3. Cognition

While3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc polyunsaturated fatty acid supplementation has been linked to improved cardiovascular health [31], the body of published clinical trials on the potential cognitive benefits that may result from improving 3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc fatty acid intakes in healthy young adults is limited. 3b2:?thyc=10Omega-33b2:?thyc polyunsaturated fatty acids may improve brain functions by facilitating vasodilatation and perfusion [32]. DHA supplementation in subjects with very low dietary intakes of DHA and EPA showed significant improvement in working memory, completing a working memory task 20% faster relative to placebo [33]. A study investigating which 3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc polyunsaturated fatty acid, 3b2:?thyc=10EPA-rich3b2:?thyc or 3b2:?thyc=10DHA-rich,3b2:?thyc might improve cognitive performance and functional brain activation in young adults showed that 3b2:?thyc=10EPA-rich3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc fatty acids showed greater cognitive performance in a Stroop 3b2:?thyc=10color-word3b2:?thyc test [34]. While krill oil is a 3b2:?thyc=10EPA-rich3b2:?thyc source of 3b2:?thyc=10omega-33b2:?thyc fatty acids, the completion time in the Stroop 3b2:?thyc=10color-word3b2:?thyc test improved after the supplementation period in the krill oil group as well as in the control subjects. This may be due to a familiarization effect. The participants knew the situation and tasks during the second trial and, therefore, may have attained better results.


## 4.1. mTOR Activation

Previous studies have shown that3b2:?thyc=10soy-derived3b2:?thyc phosphatidic acid (PA), 3b2:?thyc=10lyso-PA,3b2:?thyc or phosphatidylserine (PS) can stimulate a robust increase in mTOR signaling; however, 3b2:?thyc=10soy-derived3b2:?thyc PC was not effective [21]. While 3b2:?thyc=10egg-derived3b2:?thyc PA increases mTOR signaling, its effects are inferior to 3b2:?thyc=10soy-derived3b2:?thyc PA. 3b2:?thyc=10Egg-derived3b2:?thyc PA has a different fatty acid composition in comparison to 3b2:?thyc=10soy-derived3b2:?thyc PA, containing 3b2:?thyc=10omega-33b2:?thyc fatty acids, such as DHA or EPA, indicating that the fatty acid composition of the phospholipid influences mTOR signaling and 3b2:?thyc=10omega-3s3b2:?thyc likely attenuates the stimulatory effect. Krill oil is a complex mixture of different ingredients with PC being the main phospholipid. Krill 3b2:?thyc=10oil-PC,3b2:?thyc like 3b2:?thyc=10egg-derived3b2:?thyc phospholipids, is rich in 3b2:?thyc=10omega-33b2:?thyc fatty acids which suggest that other ingredients in krill oil such as PA, PS, or even astaxanthin or the combination of these ingredients, might be responsible for the effect on mTOR signaling, as astaxanthin has recently been shown to increase mTOR expressions in mice [25].3b2:?A3B2 tpb -6pt


## 4.2.3b2:?A3B2 tlsb=-0.36ptBody Composition and Athletic Performance

Phospholipids, including PC, PS, or PA, have previously been shown to improve athletic performance [21,[tnq_nbsp] 26]. PS has been shown to reduce 3b2:?thyc=10exercise-induced3b2:?thyc increases in the catabolic hormone cortisol [27] and reduce muscle soreness [28]. PA enhances 3b2:?thyc=10exercise-stimulated3b2:?thyc increases in lean body mass through activation of the mTOR pathway [21], and PC has been shown to maintain choline concentration during exercise [29]. Muscle contractions are induced by signals carried along cholinergic nerves to the muscle fiber, and strenuous exercise has been reported to result in decreased choline concentrations, which negatively influence the rates in which the neurotransmitter acetylcholine is synthesized and released. Maintaining choline levels during exercise has been linked to an increase in athletic performance. 3 g of krill oil provides 1.3 g of phospholipids. The phospholipids in krill oil consist of 90.5% choline containing phospholipids (76.1% PC, 7.0% alkyl acyl PC, 6.5% 3b2:?thyc=10lyso-PC,3b2:?thyc and 0.9% 3b2:?thyc=10lyso-alkyl3b2:?thyc acyl PC), 8.3% ethanolamine containing phospholipids (3.2% phosphatidylethanolamine (PE), 2.3% cardiolipin n-3b2:?show $132#acetyl PE 3b2:?thyc=10(CL/NAPE), 3b2:?thyc1.9% alkyl acyl PE, 0.7% 3b2:?thyc=10lyso-PE,3b2:?thyc and 0.2% 3b2:?thyc=10lyso-alkyl3b2:?thyc acyl PE), 0.7% phosphatidylinositol (PI), and 0.6% PS [30]. The 1.2 g phosphatidylcholine from 3 g of krill oil provides 160 mg choline, and krill oil contains 7.8 mg 3b2:?A3B2 tlsb -.1ptPS and 0.54 mg of astaxanthin. The concentration of all individual active ingredients in 3 g krill oil is lower than the currently established individual effective dose (600 mg of PS to lower cortisol levels, 4 mg of astaxanthin, or 0.2 g per kg body weight of PC). A potential synergy of the different active ingredients at low doses could not be established in this trial.While lean body mass significantly increased in the krill oil group (+1.4 kg, +2.1%) we observed no statistically significant difference in comparison to the control group (+0.3 kg, +0.5%). The increase in lean body mass was matched by an increase in muscle strength in the krill oil group (bench press +4.3 kg (+4.4%); leg press: +48.9 kg (+15.8%)); however, the increase was no different from the control group (+3.4 kg (+3.3%); leg press: +44.2 kg (+14.6%)). Perceived recovery significantly increased in the krill oil group (+15%) at the end of the trial in comparison to baseline; however, the results were not statistically different from the control group (+14%). Perceived recovery was the lowest in both groups in week 6, and subjects seem to adapt to recovering to the training stimulus by the end of the study. While the training protocol was challenging enough to see significant increases in strength, the training stimulus or the recovery periods might not have been challenging enough to elicit differences in perceived recovery. Our results contrast with previous studies showing significant improvements of postexercise muscle soreness with3b2:?thyc=10omega-33b2:?thyc supplementation [4–3b2:?show $132#8], which might be due to the lower dose of 3b2:?thyc=10omega-3s3b2:?thyc administered in our study, 963 mg for 8 weeks versus 3b2:?A3B2 tlsb -.1pt2.4–3b2:?show $132#6 3b2:?thyc=10g/d 3b2:?thycfor 1 to 8 weeks, or the difference in training stimulus and recovery periods. Based on a post hoc power analysis the sample size needed to reach statistical significance would be between 34 and 84 subjects for changes in lean body mass, depending on the level of statistical power (3b2:?thyc=10i.3b2:?show $132#e.,3b2:?thyc[tnq_nbsp] 50% to 90% probability of detecting a significant difference at an alpha level of 0.05). Future studies should investigate the activation of mTOR in vivo through a muscular biopsy.3b2:?A3B2 tpb 1pt


## 4.3. Cognition

While3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc polyunsaturated fatty acid supplementation has been linked to improved cardiovascular health [31], the body of published clinical trials on the potential cognitive benefits that may result from improving 3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc fatty acid intakes in healthy young adults is limited. 3b2:?thyc=10Omega-33b2:?thyc polyunsaturated fatty acids may improve brain functions by facilitating vasodilatation and perfusion [32]. DHA supplementation in subjects with very low dietary intakes of DHA and EPA showed significant improvement in working memory, completing a working memory task 20% faster relative to placebo [33]. A study investigating which 3b2:?thyc=10long-chain3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc polyunsaturated fatty acid, 3b2:?thyc=10EPA-rich3b2:?thyc or 3b2:?thyc=10DHA-rich,3b2:?thyc might improve cognitive performance and functional brain activation in young adults showed that 3b2:?thyc=10EPA-rich3b2:?thyc 3b2:?thyc=10omega-33b2:?thyc fatty acids showed greater cognitive performance in a Stroop 3b2:?thyc=10color-word3b2:?thyc test [34]. While krill oil is a 3b2:?thyc=10EPA-rich3b2:?thyc source of 3b2:?thyc=10omega-33b2:?thyc fatty acids, the completion time in the Stroop 3b2:?thyc=10color-word3b2:?thyc test improved after the supplementation period in the krill oil group as well as in the control subjects. This may be due to a familiarization effect. The participants knew the situation and tasks during the second trial and, therefore, may have attained better results.


## 5. Conclusion

Krill oil activates mTOR signaling. Krill oil supplementation in athletes is safe, and while no significant effects on cognition and strength were observed, its effects on body composition in combination with resistance exercise deserves further research.



---

*Source: 7625981-2018-04-26.xml*