Hematological and Hemodynamic Responses to Acute and Short-Term Creatine Nitrate Supplementation

nutrients

Article

Hematological and Hemodynamic Responses to Acute

and Short-Term Creatine Nitrate Supplementation

Ryan L. Dalton

, Ryan J. Sowinski

, Tyler J. Grubic

, Patrick B. Collins

, Adriana M. Coletta

Aimee G. Reyes

, Brittany Sanchez

, Majid Koozehchian

, Yanghoon P. Jung

Christopher Rasmussen

, Mike Greenwood

, Peter S. Murano

, Conrad P. Earnest

1,3

and Richard B. Kreider

Exercise and Sport Nutrition Lab, Human Clinical Research Facility, Texas A & M University,

College Station, TX 77843-4253, USA; [email protected] (R.L.D.);

[email protected] (R.J.S.); [email protected] (T.J.G.); [email protected] (P.B.C.);

[email protected] (A.M.C.); agr[email protected] (A.G.R.); [email protected] (B.S.);

[email protected] (M.K.); [email protected] (Y.P.J.); [email protected] (C.R.);

[email protected] (M.G.); [email protected] (C.P.E.)

Institute for Obesity and Program Evaluation, Texas A & M University, College Station, TX 77843, USA;

[email protected]

Clinical Science Division, Nutrabolt, 3891 S. Traditions Drive, Bryan, TX 77807, USA

* Correspondence: [email protected]; Tel.: +1-979-458-1498

Received: 14 November 2017; Accepted: 4 December 2017; Published: 15 December 2017

Abstract:

In a double-blind, crossover, randomized and placebo-controlled trial; 28 men and women

ingested a placebo (PLA), 3 g of creatine nitrate (CNL), and 6 g of creatine nitrate (CNH) for

6 days. Participants repeated the experiment with the alternate supplements after a 7-day washout.

Hemodynamic responses to a postural challenge, fasting blood samples, and bench press, leg press,

and cycling time trial performance and recovery were assessed. Data were analyzed by univariate,

multivariate, and repeated measures general linear models (GLM). No signiﬁcant differences were

found among treatments for hemodynamic responses, clinical blood markers or self-reported side

effects. After 5 days of supplementation, one repetition maximum (1RM) bench press improved

signiﬁcantly for CNH (mean change, 95% CI; 6.1 [3.5, 8.7] kg) but not PLA (0.7 [

−

1.6, 3.0] kg or

CNL (2.0 [

−

0.9, 4.9] kg, CNH, p = 0.01). CNH participants also tended to experience an attenuated

loss in 1RM strength during the recovery performance tests following supplementation on day 5

(PLA:

−

9.3 [

−

13.5,

−

5.0], CNL:

−

9.3 [

−

13.5,

−

5.1], CNH:

−

3.9 [

−

6.6,

−

1.2] kg, p = 0.07). After 5 days,

pre-supplementation 1RM leg press values increased signiﬁcantly, only with CNH (24.7 [8.8, 40.6] kg,

but not PLA (13.9 [

−

15.7, 43.5] or CNL (14.6 [

−

0.5, 29.7]). Further, post-supplementation 1RM

leg press recovery did not decrease signiﬁcantly for CNH (

−

13.3 [

−

31.9, 5.3], but did for PLA

(

−

30.5 [

−

53.4,

−

7.7] and CNL (

−

29.0 [

−

49.5,

−

8.4]). CNL treatment promoted an increase in bench

press repetitions at 70% of 1RM during recovery on day 5 (PLA: 0.4 [

−

0.8, 1.6], CNL: 0.9 [0.35, 1.5],

CNH: 0.5 [

−

0.2, 0.3], p = 0.56), greater leg press endurance prior to supplementation on day 5

(PLA:

−

0.2 [

−

1.6, 1.2], CNL: 0.9 [0.2, 1.6], CNH: 0.2 [

−

0.5, 0.9], p = 0.25) and greater leg press

endurance during recovery on day 5 (PLA:

−

0.03 [

−

1.2, 1.1], CNL: 1.1 [0.3, 1.9], CNH: 0.4 [

−

0.4, 1.2],

p = 0.23). Cycling time trial performance (4 km) was not affected. Results indicate that creatine nitrate

supplementation, up to a 6 g dose, for 6 days, appears to be safe and provide some ergogenic beneﬁt.

Keywords: creatine; nitrate; safety; dietary supplement; ergogenic aid

1. Introduction

Creatine has proven to be a safe and effective nutritional ergogenic aid for improving high intensity

exercise performance [

–

]. The primary form of creatine studied to date is creatine monohydrate [

Nutrients 2017, 9, 1359; doi:10.3390/nu9121359 www.mdpi.com/journal/nutrients

Nutrients 2017, 9, 1359 2 of 22

Short-term creatine monohydrate supplementation (i.e., 0.3 g/kg/day for 3–7 days) and/or long-term

creatine supplementation (e.g., 0.03 g/kg/day) has been reported to increase muscle phosphocreatine

levels by 10–40%, improve high-intensity exercise performance, and enhance training adaptations,

leading to an increase in muscle mass [

–

]. More recently, a number of clinical applications of

creatine supplementation have been studied involving neurodegenerative diseases (e.g., muscular

dystrophy, Parkinson’s, Huntington’s disease), diabetes, osteoarthritis, ﬁbromyalgia, aging, brain and

heart ischemia, adolescent depression, and pregnancy [

]. Collectively, these studies provide a large

body of evidence that creatine can not only improve exercise performance, but can play a role in

preventing and/or reducing the severity of injury, enhancing rehabilitation from injuries, and helping

athletes tolerate heavy training loads [

]. For this reason, creatine is one of the most common

nutrients found in dietary supplements marketed to active individuals and it is increasingly being

recommended as a dietary strategy to support general health as one ages [1,3,16].

Over the years, a number of different forms of creatine have been introduced into the market,

each purported to promoted greater bioavailability and/or efﬁcacy [

]. Our group has evaluated the

efﬁcacy and safety of a number of newer forms of creatine, including serum creatine [

], effervescent

creatine [

], creatine ethyl ester [

], and a buffered form of creatine [

]. These studies have

shown that while these forms of creatine may offer some beneﬁts, they do not appear to promote

greater adaptations than creatine monohydrate [

]. Additionally, we have investigated whether

co-ingestion of creatine with carbohydrate [

], protein [

], carbohydrate and protein weight gain

supplements [

], D-pinitol [

], fenugreek extract [

], beta alanine [

], and Russian Tarragon [

]

enhance the bioavailability and/or beneﬁts than creatine supplementation alone. These studies have

generally indicated that co-ingesting creatine with other nutrients that have been shown to enhance

creatine retention, exercise capacity, and/or training adaptations can provide synergistic and/or

additive beneﬁts.

More recently, nitrate supplementation, primarily in the form of beetroot juice or inorganic nitrates,

has been reported to improve exercise capacity [

–

]. The ergogenic dose of nitrates is about 300 mg,

ingested 1 to 2 h prior to exercise or for several days prior to performance [

]. For example, Lansley

and colleagues [

] reported that acute dietary nitrate supplementation increases 4- and 16.1-km

cycling time trial performance. Clifford and coworkers [

] reported that beetroot juice ingestion

reduced the decrement in jumping performance and reactive strength index following repeated sprint

training. Mosher et al. [

] reported that nitrate supplementation (400 mg) increased the number of

repetitions performed to failure and total work performed during resistance training. Finally, beetroot

ingestion has been reported to enhance repetitive sprint performance [

]. For this reason, a number

of dietary supplements are now available containing beetroot and/or inorganic nitrates. Additionally,

it has been recommended that individuals consume a diet high in nitrates (e.g., celery, cress, lettuce,

red beetroot, spinach, rucola) with a goal of ingesting 1200 mg/day or more of nitrates as a means of

managing hypertension and/or reducing risk to cardiovascular disease [42,43].

Creatine nitrate (i.e., C

) was introduced to the market about six years ago. It is generally

formed by combining nitric acid and creatine with water until crystallized into creatine dinitrate

or creatine trinitrate [

]. Alternatively, it can be formed using nitrous acid instead of nitric acid

and yielding creatine nitrate [

]. The rationale in combining creatine with nitrate was to enhance

vasodilation and thereby theoretically enhance creatine absorption and/or efﬁcacy [

]. While creatine

and nitrate have been studied independently, less is known about the effects of creatine nitrate

supplementation on exercise performance and/or recovery and some have expressed concerns over

potential health risks [

–

]. Joy and colleagues [

] reported that ingestion of 1 or 2 g/day of creatine

nitrate for 28 days appeared to be safe in healthy individuals. Our group recently reported that acute

ingestion of 1.5 g and 3 g of creatine nitrate did not increase muscle creatine content as much as creatine

monohydrate or affect acute heart rate, blood pressure, or hematological responses [

]. Additionally,

28 days of creatine nitrate supplementation during training (i.e., 6 or 12 g/day for 7 days and 1.5 g or

3 g/day for 21 days) did not increase muscle creatine levels to the same degree as creatine monohydrate.

Nutrients 2017, 9, 1359 3 of 22

However, some performance advantages were seen in comparison to creatine monohydrate, apparently

due to increased nitrate availability. We also evaluated the acute (one dose) and chronic (28 days)

effects of ingesting a pre-workout supplement containing 2 g per serving of creatine nitrate as part of

a pre-workout supplement [

]. These studies did not identify any signiﬁcant negative side effects

from acute or chronic creatine nitrate supplementation. However, more research is needed, particularly

at the higher doses that some athletes may take, to examine safety and efﬁcacy.

The purpose of this study was to examine the acute and short-term effects (0, 1, 5 and 6 days)

of ingesting 3 g and 6 g doses of creatine nitrate on hemodynamic responses to a postural challenge,

prior to and following, exercise, exercise performance, and recovery. The primary outcome was an

assessment of safety, as determined by evaluating hemodynamic responses to a postural challenge,

prior to and following, intense exercise, fasting hematology, and self-reported symptoms and side

effects. The secondary outcome was an assessment of performance, as determined by examining upper

and lower body one repetition maximum (1RM), muscular endurance, and 4 km cycling time trial

performance. We hypothesized that acute and short-term creatine nitrate would enhance performance

and/or recovery, while posing no adverse side effects.

2. Methods

2.1. Experimental Design

Prior to starting the study, approval was obtained from the Texas A & M University Institutional

Review Board (IRB2015-0684F). This study is also registered with clinicatrials.gov (#NCT03039829).

This study was conducted at a university-based research setting in a double-blind, randomized,

counter-balanced, crossover, and repeated measures manner, after obtaining informed consent from

each participant. The independent variable was nutritional supplementation. Figure 1 shows the order

of tests performed for each experiment, while Figure 2 presents the order of testing on each testing

day. The study design allowed for the assessment of acute and short-term responses to creatine nitrate

supplementation on performance and recovery from resistance exercise. The following describes the

general methods employed.

Nutrients 2017, 9, 1359 3 of 22

[52]. Additionally, 28 days of creatine nitrate supplementation during training (i.e., 6 or 12 g/day for

7 days and 1.5 g or 3 g/day for 21 days) did not increase muscle creatine levels to the same degree as

creatine monohydrate. However, some performance advantages were seen in comparison to creatine

monohydrate, apparently due to increased nitrate availability. We also evaluated the acute (one dose)

and chronic (28 days) effects of ingesting a pre-workout supplement containing 2 g per serving of

creatine nitrate as part of a pre-workout supplement [53,54]. These studies did not identify any

significant negative side effects from acute or chronic creatine nitrate supplementation. However,

more research is needed, particularly at the higher doses that some athletes may take, to examine

safety and efficacy.

The purpose of this study was to examine the acute and short-term effects (0, 1, 5 and 6 days) of

ingesting 3 g and 6 g doses of creatine nitrate on hemodynamic responses to a postural challenge,

prior to and following, exercise, exercise performance, and recovery. The primary outcome was an

assessment of safety, as determined by evaluating hemodynamic responses to a postural challenge,

prior to and following, intense exercise, fasting hematology, and self-reported symptoms and side

effects. The secondary outcome was an assessment of performance, as determined by examining

upper and lower body one repetition maximum (1RM), muscular endurance, and 4 km cycling time

trial performance. We hypothesized that acute and short-term creatine nitrate would enhance

performance and/or recovery, while posing no adverse side effects.

2. Methods

2.1. Experimental Design

Prior to starting the study, approval was obtained from the Texas A & M University Institutional

Review Board (IRB2015-0684F). This study is also registered with clinicatrials.gov (#NCT03039829).

This study was conducted at a university-based research setting in a double-blind, randomized,

counter-balanced, crossover, and repeated measures manner, after obtaining informed consent from

each participant. The independent variable was nutritional supplementation. Figure 1 shows the

order of tests performed for each experiment, while Figure 2 presents the order of testing on each

testing day. The study design allowed for the assessment of acute and short-term responses to

creatine nitrate supplementation on performance and recovery from resistance exercise. The

following describes the general methods employed.

Figure 1. Overview of study design.

Nutrients 2017, 9, 1359 4 of 22

Figure 2. Testing sequence timeline. (A) Presents the testing sequence on days 0 and 5, while

(B) shows the testing sequence on days 1 and 6 of supplementation.

2.2. Participants

Figure 3 presents a CONSORT diagram. Apparently healthy and recreationally active men and

women, between the ages 18–40 years, were recruited to participate in this study. Individuals who

expressed interest from email or study advertisements were interviewed to determine if they met

initial screening eligibility to participate in this study. Those who met initial screening qualifications,

were invited to attend a familiarization session in which they received written and verbal

explanations of the study design, and testing procedures, and were able to review the informed

consent document. Interested individuals signed the informed consent and then completed personal

and medical histories and had height, weight, resting blood pressure, and heart rate measured. A

registered nurse reviewed medical history forms and physical examination measurements to

determine eligibility to participate. Inclusion criteria required that each participant have at least 6

months of resistance training immediately prior to entering the study, inclusive of performing bench

press and leg press or squats. Participants were excluded from participation if they had a history of

treatment for metabolic disease (i.e., diabetes), hypertension, hypotension, thyroid disease,

arrhythmias, and/or cardiovascular disease; they were currently using any prescription medication

(birth control was allowed); they were a pregnant or lactating female or planned to become pregnant

within the next month; they had a history of smoking; they drank excessively (12 drinks per week);

or they had a recent history of creatine or nitrate supplementation within eight weeks of the start of

supplementation. Participants were 21.6 ± 3.7 years of age, 172.1 ± 8.2 cm tall, and weighed 73.4 ± 10.9

kg.

Figure 2.

Testing sequence timeline. (

) Presents the testing sequence on days 0 and 5, while (

) shows

the testing sequence on days 1 and 6 of supplementation.

2.2. Participants

Figure 3 presents a CONSORT diagram. Apparently healthy and recreationally active men and

women, between the ages 18–40 years, were recruited to participate in this study. Individuals who

expressed interest from email or study advertisements were interviewed to determine if they met

initial screening eligibility to participate in this study. Those who met initial screening qualiﬁcations,

were invited to attend a familiarization session in which they received written and verbal explanations

of the study design, and testing procedures, and were able to review the informed consent document.

Interested individuals signed the informed consent and then completed personal and medical histories

and had height, weight, resting blood pressure, and heart rate measured. A registered nurse

reviewed medical history forms and physical examination measurements to determine eligibility

to participate. Inclusion criteria required that each participant have at least 6 months of resistance

training immediately prior to entering the study, inclusive of performing bench press and leg press or

squats. Participants were excluded from participation if they had a history of treatment for metabolic

disease (i.e., diabetes), hypertension, hypotension, thyroid disease, arrhythmias, and/or cardiovascular

disease; they were currently using any prescription medication (birth control was allowed); they were

a pregnant or lactating female or planned to become pregnant within the next month; they had a history

of smoking; they drank excessively (12 drinks per week); or they had a recent history of creatine

or nitrate supplementation within eight weeks of the start of supplementation. Participants were

21.6 ± 3.7 years of age, 172.1 ± 8.2 cm tall, and weighed 73.4 ± 10.9 kg.

Nutrients 2017, 9, 1359 5 of 22

Figure 3. CONSORT diagram.

2.3. Familiarization

Participants who met eligibility criteria and were cleared to participate in the study underwent

a familiarization session in which Dual-Energy X-ray Absorptiometry determined body composition

and bioelectrical impedance (BIA) determined body water measurements were obtained.

Additionally, participants completed 1RM muscular strength and 70% of 1RM muscular endurance

tests on the bench press and leg press. This was followed by warming up on a cycle ergometer and

performing a 4 km cycle ergometer time trial. These tests served to familiarize the participants with

the testing procedures and served as baseline performance comparators. After exercise testing was

completed, participants scheduled their lab visits (4 testing sessions per week of each treatment

experiment followed by a 7-day washout period between experiments).

2.4. Supplementation Protocol

Participants were assigned in a randomized, counter balanced, double-blind, and cross-over

manner, to one of three supplement treatments for each testing week, while following their normal

diet. The supplements consisted of: (1.) 6.0 g of a dextrose placebo (PLA); 3.0 g of creatine nitrate (2 g

creatine; 1 g nitrate, CNL) with 3.0 g of dextrose; or, (2.) 6.0 g of creatine nitrate (4 g creatine; 2 g

nitrate, CNH). Nutrabolt (Bryan, TX, USA) provided all of the supplements for this study.

Supplements were prepared and packaged by Thermo-life International (Phoenix, AZ, USA). All

supplements were provided in identical clear plastic packets, with the only differentiating

characteristic being the letter A, B, or C printed on the label of each packet. All supplements were

indistinguishable from each other, based on taste, texture, and appearance. Supplements were

administered in a double-blinded manner. Participants were instructed to mix the entire contents of

the packet with 16 ounces of water. Compliance was monitored at each testing session by

administering the supplements and observing ingestion as well as from side effect questionnaires.

During each 6-day experiment, supplements were administered after completing the first bout

of exercise on day 0. On day 1, the second supplement dose was consumed after donating blood in a

fasted state. Participants were given 3 packets of supplements after completing testing on day 1 and

were instructed to ingest one packet, mixed with water, each day for the next three days (Days 2, 3,

and 4). On day 5, participants ingested the sixth dose of their assigned supplement, immediately

following the first round of exercise. On day 6, participants consumed their seventh and final dose of

Figure 3. CONSORT diagram.

2.3. Familiarization

Participants who met eligibility criteria and were cleared to participate in the study underwent

a familiarization session in which Dual-Energy X-ray Absorptiometry determined body composition

and bioelectrical impedance (BIA) determined body water measurements were obtained. Additionally,

participants completed 1RM muscular strength and 70% of 1RM muscular endurance tests on the bench

press and leg press. This was followed by warming up on a cycle ergometer and performing a 4 km

cycle ergometer time trial. These tests served to familiarize the participants with the testing procedures

and served as baseline performance comparators. After exercise testing was completed, participants

scheduled their lab visits (4 testing sessions per week of each treatment experiment followed by a 7-day

washout period between experiments).

2.4. Supplementation Protocol

Participants were assigned in a randomized, counter balanced, double-blind, and cross-over

manner, to one of three supplement treatments for each testing week, while following their normal diet.

The supplements consisted of: (1.) 6.0 g of a dextrose placebo (PLA); 3.0 g of creatine nitrate (2 g creatine;

1 g nitrate, CNL) with 3.0 g of dextrose; or, (2.) 6.0 g of creatine nitrate (4 g creatine; 2 g nitrate, CNH).

Nutrabolt (Bryan, TX, USA) provided all of the supplements for this study. Supplements were

prepared and packaged by Thermo-life International (Phoenix, AZ, USA). All supplements were

provided in identical clear plastic packets, with the only differentiating characteristic being the letter

A, B, or C printed on the label of each packet. All supplements were indistinguishable from each

other, based on taste, texture, and appearance. Supplements were administered in a double-blinded

manner. Participants were instructed to mix the entire contents of the packet with 16 ounces of water.

Compliance was monitored at each testing session by administering the supplements and observing

ingestion as well as from side effect questionnaires.

During each 6-day experiment, supplements were administered after completing the ﬁrst bout of

exercise on day 0. On day 1, the second supplement dose was consumed after donating blood in a fasted

state. Participants were given 3 packets of supplements after completing testing on day 1 and were

instructed to ingest one packet, mixed with water, each day for the next three days (Days 2, 3, and 4).

On day 5, participants ingested the sixth dose of their assigned supplement, immediately following the

ﬁrst round of exercise. On day 6, participants consumed their seventh and ﬁnal dose of their respective

supplement, immediately after donating a blood sample in a fasted state. Thus, participants consumed

Nutrients 2017, 9, 1359 6 of 22

7 doses of their assigned supplement (1 per day) over a 6-day period, followed by a 7-day washout

period where no supplement was consumed, nor testing performed. After this, the supplementation

and testing schedule was repeated with the participants’ next assigned supplement. This schedule

was repeated a total of 3 times (Figure 1), in a counterbalanced and double-blind manner.

2.5. Testing Sequence

Participants arrived at the laboratory on days 0, 1, 5, and 6 during each treatment period.

Participants were instructed to refrain from exercise, alcohol, and non-steroidal anti-inﬂammatory

drugs (NSAID) consumption for 48 h prior to each testing session. All fasting blood samples were

obtained following an 8 h fast, primarily between the hours of 6:00–9:00 a.m. On days 0 and 5

(Figure 2A), participants donated a venous blood sample and completed a pre-exercise side effects

questionnaire. Participants were then weighed and BIA total body water measurements were obtained.

Participants then performed a pre-supplementation hemodynamic postural challenge test, using a tilt

table, which consisted of obtaining heart rate and blood pressure measurements in supine and vertical

positions, prior to and following a postural challenge. Subjects then performed 1RM tests on the bench

press and leg press, followed by performing 3 sets of 10 repetitions at 70% of 1RM, with the last set to

failure. Participants then ingested their assigned supplement and waited 15 min before repeating the

hemodynamic reactivity test. Approximately 30 min after ingestion of the supplement, participants

repeated 1RM tests on the bench press and leg press. This was followed by performing 1 set to failure

at 70% of 1RM on the bench press and leg press. The rationale for this approach was to determine

whether ingestion of the supplements would inﬂuence exercise capacity after exhaustive exercise and

toward the end of a training session. Participants then completed a side effects questionnaire.

On days 1 and 6 (Figure 2B), participants reported to the lab at approximately the same time of

day following an 8 h fast and donated a fasting blood sample. This was followed by completing a side

effects questionnaire. The subject then ingested one dose of their assigned supplement. Approximately

30 min after ingesting the supplement, the participant warmed up and performed a 4 km cycling time

trial on an electronically-braked ergometer. Participants were encouraged to complete the 4 km distance

as quickly as possible. After completing the time trial, participants completed a post exercise side

effects questionnaire. Participants observed a 7-day washout period before repeating the experiment

while consuming the alternate treatments.

3. Procedures

3.1. Anthropometry & Body Composition

Standardized anthropological testing included assessments for body mass and height,

on a Healthometer Professional 500KL (Pelstar LLC, Alsip, IL, USA) self-calibrating digital scale,

with an accuracy of

0.02 kg. Total body water was determined under standardized conditions,

using an ImpediMed DF50 BIA analyzer (ImpediMed, San Diego, CA, USA). Whole body bone density

and body composition measures (excluding cranium) were determined with a Hologic Discovery W

DEXA (Hologic Inc., Waltham, MA, USA) equipped with APEX Software (APEX Corporation Software,

Pittsburg, PA, USA) by using standardized procedures [

]. On the day of each test, the equipment

was calibrated following the manufacturer’s guidelines.

3.2. Hemodynamic Challenge Assessment

Participants were placed on a standard tilt table in a supine position (IRONMAN Gravity 4000

Inversion Table; Paradigm Health & Wellness, Inc., City of Industry, CA, USA). After 15 min of

motionless rest, heart rate was taken at the radial artery and systolic and diastolic blood pressure was

measured by listening for Korotkoff sounds from the brachial artery at the antecubital area of the elbow

using standard stethoscopes and sphygmomanometers. Next, the tilt table was adjusted to vertical,

where the participant rested for 2 min and the metrics were re-assessed. Mean arterial pressure was

Nutrients 2017, 9, 1359 7 of 22

calculated as 1/3 systolic blood pressure plus 2/3 diastolic blood pressure [

]. Pulse pressure was

calculated as the difference between systolic and diastolic blood pressure [

]. Rate pressure product

was calculated as heart rate multiplied by systolic blood pressure [59].

3.3. Blood Collection Procedures

Participants provided an 8 h fasted blood sample via venipuncture of an antecubital vein in

the forearm, in accordance with standard phlebotomy procedures. Approximately 10 mL of whole

blood was collected at the beginning of each testing day, in one 3.5 mL BD Vacutainer

K2 EDTA

tube (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and one 8.5 mL BD Vacutainer

serum separation tube (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Both tubes sat

at room temperature for 15 min, then the 8.5 mL serum separation tube was centrifuged at 3500 rpm

for 10 min using a 4

◦

C refrigerated bench top ThermoScientiﬁc Heraeus MegaFuge 40R Centrifuge

(Thermo Electron North America LLC, West Palm Beach, FL, USA). Both tubes were stored at 4

◦

C for

3 to 4 h, prior to analysis or storage. Serum was stored at

−

◦

C in polypropylene microcentrifuge

tubes for later analysis.

3.4. Blood Chemistry Analysis

A complete blood count with platelet differential (hemoglobin, hematocrit, red blood cell counts,

mean corpuscular volume (MCV), mean corpuscle hemoglobin (MCH), mean corpuscular hemoglobin

concentration (MCHC), red blood cell distribution width (RDW), white blood cell counts, lymphocytes,

granulocytes, and mid-range absolute count (MID)) was measured using a Abbott Cell Dyn 1800

(Abbott Laboratories, Abbott Park, IL, USA) automated hematology analyzer. The internal quality

control for Abbott Cell Dyn 1800 was performed using three levels of control ﬂuids purchased from

manufacturer to calibrate acceptable SD and CV values for all whole blood cell parameters. Test-to-test

reliability assessment of assays evaluated in the study yielded mean CVs of <6.3% with r values > 0.9.

Serum blood samples were analyzed for the following: glucose, alkaline phosphatase (ALP),

aspartate transaminase (AST), alanine transaminase (ALT), creatinine, blood urea nitrogen (BUN),

creatine kinase (CK), lactate dehydrogenase (LDH), glucose, total cholesterol, high density lipoprotein

(HDL), low density lipoprotein (LDL), and triglycerides (TG), using a Cobas c111 (Roche Diagnostics,

Basel, Switzerland) automated clinical chemistry analyzer. The analyzer was calibrated daily, as per the

manufacturer’s guidelines and has been shown to be valid and reliable in previously published

reports [

]. Internal quality control was performed using two levels of control ﬂuids purchased

from the manufacturer to calibrate acceptable standard deviation (SD) and coefﬁcient of variation (C

)

values for all assays. Samples were re-run if the values observed were outside control values and/or

clinical norms, according to standard procedures. A prior analysis in our lab has yielded test-to-test

reliability of a range of CVs, from 0.4 to 2.4% for low control samples and 0.6–1.9% for high controls.

Precision has been found between 0.8 and 2.4% for low controls and 0.5–1.7% for high controls.

3.5. Side Effects

Side effects questionnaires were used to document how well participants tolerated each treatment

as well as to monitor compliance to the supplementation protocol. Participants were asked to rank the

frequency and severity of dizziness, headache, tachycardia, heart skipping or palpitations, shortness of

breath, nervousness, blurred vision, and unusual or adverse effects. Participants were requested to rank

their perceived symptoms with 0 (none), 1 (minimal: 1–2/week), 2 (slight: 3–4/week), 3 (occasional:

5–6/week), 4 (frequent: 7–8/week), or 5 (severe: 9 or more/week).

3.6. Muscular Strength and Endurance Assessment

Bench press tests were performed using a standard isotonic Olympic bench press (Nebula Fitness,

Versailles, OH, USA), while leg press was determined using a hip/leg sled (Nebula Fitness, Versailles,

OH, USA), using standard procedures [

]. Participants performed three warm up sets, prior to

Nutrients 2017, 9, 1359 8 of 22

performing 1RM attempts (i.e., one set of 10 at 50%, one set of 5 at 70%, and one set of 3 at 90%

of anticipated 1RM). Following determination of 1RM outcomes, participants performed two sets

of 10 repetitions with 2 min rest and recovery between sets, at the closest bar/leg press weight

corresponding to 70% of familiarization session 1RM. Participants then rested for 2 min before

performing a third set to failure. After 2 min of rest, participants followed the same procedure

to determine leg press 1RM and leg press muscular endurance. Hand placement on the bench press

bar and seat and foot positioning on the leg press were in the same position for all attempts and

testing sessions. Test-to-test reliability of performing these tests in our lab on resistance-trained

participants has yielded low C

s and high reliability for the bench press (1.9%, r = 0.94) and hip

sled/leg press (0.7%, r = 0.91).

The initial strength tests were performed to pre-fatigue the participant before assessing recovery

performance after supplement ingestion. The recovery muscular strength and endurance performance

assessment involved performing a 1RM test and then one set to failure at 70% of the familiarization

1RM, following similar procedures as those described above. In this way, the effects of acute

and short-term supplementation could be assessed on muscular strength and endurance recovery.

Total 1RM weight lifted in kg and the number repetitions performed for each set using 70% of

the familiarization weight (rounded to the nearest 2.27 kg or 5 Lbs. that could be put on the bar)

were recorded. Total lifting volume was calculated by multiplying the 70% of 1RM weight lifted by the

number of repetitions performed each set. Day-to-day test reliability for performing this performance

test in our lab on resistance-trained participants has yielded a CV of 0.34 and an intraclass correlation

coefﬁcient of 0.99 for three sets of bench press total lifting volume and an intraclass correlation

coefﬁcient of 0.96 for three sets of leg press total lifting volume.

3.7. Cycling Time Trial Performance Assessment

Time trial performance was examined on a magnetically-braked cycle ergometer (Lode Sport

Excalibur Sport 925900, Groningen, The Netherlands) over a distance of 4 km. The seat height,

seat position, handlebar height, and handlebar position were recorded for each participant to use for

each testing session. The test began with a 3-min warm-up, comprised of pedaling against a resistance

of 25 W for the ﬁrst minute, 50 W for the second minute, and 100 W for the third minute. At the

completion of the warm up, a standardized resistance (4 J/kg/rev) was applied and the participant was

instructed to complete the distance the shortest time possible. Upon completion, the participant was

instructed to continue at a slow pace to facilitate recovery. Data were recorded as time to completion

and average power in Watts. Mean test-retest reliability studies performed in our lab over repeated

days revealed mean CVs for time to completion of 0.235, with a mean intraclass correlation of 0.850.

3.8. Statistical Analysis

Data were analyzed using IBM

SPSS

Version 24 software (IBM Corp., Armonk, NY, USA).

The sample size was determined based on the expectation of a ﬁve percent improvement in exercise

performance and corresponding power of 0.80 as well as prior research in our lab using a similar

research design [

]. Baseline demographic data were analyzed using one-way ANOVA. Data were

examined for a treatment order effect to conﬁrm that randomization procedures were effective.

Data were analyzed using univariate, multivariate and repeated measures general linear models (GLM),

with and without gender as a covariate. Since results were consistent, results are reported without the

covariate included. Wilks’ Lambda multivariate tests are reported to describe overall effects of related

variables analyzed. Greenhouse–Geisser univariate tests with least signiﬁcant difference post-hoc

comparisons are presented for individual variables analyzed. Hematological variables were also

examined relative to normal clinical limits, to examine the frequency of changes in hematology outside

of normal, clinical limits, from baseline to follow-up, using Chi-square and adjusted residual analyses.

These analyses examined the likelihood of excursions outside of clinical limits for each treatment, as

follows: (1) no change; (2) normal at baseline, high at follow-up; (3) high at baseline, high at follow-up;

Nutrients 2017, 9, 1359 9 of 22

(4) high at baseline, normal at follow-up. Delta changes (post–pre) were calculated on selected variables

and analyzed by one-way ANOVA with Sidak post-hoc analyses. Data are reported as mean (SD),

mean change from baseline (95% CI), and frequency of occurrence, according to the Chi-square analysis.

Data were considered statistically signiﬁcant when the probability of type I error was 0.05 or less,

while tendencies towards statistical signiﬁcance were noted when p-levels were p > 0.05 to p < 0.10.

Partial eta squared effect sizes (

) were also reported on select variables as an indicator of effect size.

An eta squared of around 0.02 was considered small, 0.13 medium, and 0.26 large. Primary outcomes

included various indices of hematologic and hemodynamic changes accompanying supplementation.

Secondary outcomes included various indices of exercise performance. Mean changes with 95% CIs

completely above or below baseline were considered signiﬁcantly different [62].

4. Results

4.1. Baseline Characteristics

A total of 38 individuals met the initial screening criteria and consented to participate in this study

(Figure 3). Four participants declined to participate after undergoing familiarization assessments.

Therefore, 34 individuals were allocated into treatments to begin the cross-over study. Six participants

did not complete the entire study due to illness unrelated to the treatment: family emergency, and

time constraints. Statistical analyses were performed on 28 individuals (18 men and 10) women

(Table 1). No signiﬁcant differences were observed among gender in age and body mass indices.

However, as expected, men were taller, weighed more, had more fat free mass, and had less body fat

than females.

Table 1. Participant Characteristics.

Total Male Female p-Level

N 28 18 10

Age (years) 21.6 ± 3.7 21.4 ± 3.0 22.1 ± 4.7 0.40

Height (m) 1.72 ± 0.08 1.76 ± 0.06

1.65 ± 0.06

†

0.001

Weight (kg) 73.1 ± 11.4 76.6 ± 9.0

66.9 ± 12.6

†

0.001

Body Mass Index (BMI) (kg/m

)

24.7 ± 2.8 24.8 ± 2.9 24.5 ± 2.8 0.63

Fat Free Mass (kg) 53.5 ± 10.3 59.9 ± 6.1

41.9 ± 4.2

†

0.001

Fat Mass (kg) 13.8 ± 8.0 10.7 ± 6.2

19.6 ± 7.7

†

0.001

Body Fat (%) 20.4 ± 10.5 14.6 ± 7.0

30.9 ± 7.1

†

0.001

Data are mean ± SD. p < 0.05 is considered signiﬁcant. (

†

) denotes a signiﬁcant difference from male.

4.2. Primary Outcome—Safety

4.2.1. Hemodynamic Response

Table S1 presents data from the hemodynamic reactivity test. The multivariate analysis revealed

no signiﬁcant overall Wilks’ Lambda treatment

time (p = 0.38) or treatment

time

gender (p = 0.45)

effects among (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), pulse pressure (PP),

heart rate (HR), or rate pulse product (RPP). Similarly, no signiﬁcant univariate treatment

time or

treatment

time

gender effects were observed for SBP, DBP, MAP, PP, HR, or RPP. Figure 4 shows

mean changes with 95% CIs for SBP, DBP, and HR. No signiﬁcant changes from baseline supine values

were seen in SBP. Additionally, no signiﬁcant (p < 0.05) differences were observed among treatments

at any time point, although SBP, in the CNL treatment, tended to be lower than the CNH responses

(p = 0.095). However, neither of these values differed from the PLA treatment (PLA: 1.1 [

−

3.4, 5.7],

CNL:

−

1.2 [

−

5.9, 3.5], CNH: 2.5 [

−

0.8, 5.9] mmHg, p = 0.10). No signiﬁcant changes were observed

over time in DBP responses or among treatments at any data point. HR values signiﬁcantly increased

above pre-supplementation supine baseline values, as a result of moving to a vertical position and

Nutrients 2017, 9, 1359 10 of 22

in response to exercise. However, no signiﬁcant differences were observed among treatments at any

data point.

Nutrients 2017, 9, 1359 10 of 22

(PLA: 1.1 [−3.4, 5.7], CNL: −1.2 [−5.9, 3.5], CNH: 2.5 [−0.8, 5.9] mmHg, p = 0.10). No significant changes

were observed over time in DBP responses or among treatments at any data point. HR values

significantly increased above pre-supplementation supine baseline values, as a result of moving to a

vertical position and in response to exercise. However, no significant differences were observed

among treatments at any data point.

Nutrients 2017, 9, 1359 11 of 22

Figure 4. Systolic blood pressure (A), diastolic blood pressure (B), and heart rate (C) responses from

to the postural hemodynamic challenge test from baseline values for the placebo (PLA), low dose

creatine nitrate (CNL), and high dose creatine nitrate (CNH) treatments. Data are mean changes (95%

CI). Confidence intervals not crossing zero are statistically significant (p < 0.05). c Represents p < 0.05

difference from CNH. † Represents p > 0.05 to p < 0.05 effect.

4.2.2. Hematology Assessment

Tables S2–S8 present the results of whole blood and serum markers monitored in this study. No

significant overall multivariate or univariate treatment × time or treatment × gender × time

interactions were observed. Table S9 shows Chi square analysis of changes from baseline values

observed. No significant changes were observed among treatments. Overall, some perturbations in

blood chemistries exceeding normal clinical limits at baseline and follow-up were observed before

and after supplement ingestion. However, no significant differences were seen in the frequency of

occurrences among treatments.

4.2.3. Self-Reported Side Effects

Tables S10 and S11 present the frequency and severity of monitored side effects. Participants

were asked to rate the frequency and severity of the following eight symptoms before and after

testing each testing day: dizziness, headaches, tachycardia, heart palpitations, dyspnea, nervousness,

blurred vision, and other symptoms. No significant differences at any time point among any of the

treatments for frequency or severity of symptoms were found. Additionally, as Table S12 shows,

creatine nitrate supplementation was not associated with dehydration.

4.3. Secondary Outcome—Performance

4.3.1. Bench Press and Leg Press Performance

Table 2 presents bench and leg press muscular strength and endurance performance results.

Bench press and leg press 1RM results were analyzed in absolute (kg) and relative terms (kg/kg

FFM)

and since results were consistent, absolute values are reported. The multivariate analysis revealed

overall Wilks’ Lambda treatment (p = 0.94), time (p = 0.001), gender (p = 0.001), treatment × time (p =

0.11), treatment × gender (p = 0.90), time × gender (p = 0.001), and treatment × time × gender (p = 0.57)

effects. The univariate analysis revealed that although some time and gender effects were observed,

no significant treatment × time or gender × time effects were seen in bench press 1RM, bench press

endurance, leg press 1RM, or leg press endurance. A significant treatment × time × gender interaction

was observed in leg press endurance (p = 0.04). Post-hoc analyses revealed that leg press endurance

Figure 4.

Systolic blood pressure (

), diastolic blood pressure (

), and heart rate (

) responses from to

the postural hemodynamic challenge test from baseline values for the placebo (PLA), low dose creatine

nitrate (CNL), and high dose creatine nitrate (CNH) treatments. Data are mean changes (95% CI).

Conﬁdence intervals not crossing zero are statistically signiﬁcant (p < 0.05). c represents p < 0.05

difference from CNH. † represents p > 0.05 to p < 0.05 effect.

Nutrients 2017, 9, 1359 11 of 22

4.2.2. Hematology Assessment

Tables S2–S8 present the results of whole blood and serum markers monitored in this study.

No signiﬁcant overall multivariate or univariate treatment

time or treatment

gender

time

interactions were observed. Table S9 shows Chi square analysis of changes from baseline values

observed. No signiﬁcant changes were observed among treatments. Overall, some perturbations in

blood chemistries exceeding normal clinical limits at baseline and follow-up were observed before

and after supplement ingestion. However, no signiﬁcant differences were seen in the frequency of

occurrences among treatments.

4.2.3. Self-Reported Side Effects

Tables S10 and S11 present the frequency and severity of monitored side effects. Participants

were asked to rate the frequency and severity of the following eight symptoms before and after testing

each testing day: dizziness, headaches, tachycardia, heart palpitations, dyspnea, nervousness, blurred

vision, and other symptoms. No signiﬁcant differences at any time point among any of the treatments

for frequency or severity of symptoms were found. Additionally, as Table S12 shows, creatine nitrate

supplementation was not associated with dehydration.

4.3. Secondary Outcome—Performance

4.3.1. Bench Press and Leg Press Performance

Table 2 presents bench and leg press muscular strength and endurance performance results.

Bench press and leg press 1RM results were analyzed in absolute (kg) and relative terms (kg/kg

FFM

)

and since results were consistent, absolute values are reported. The multivariate analysis revealed

overall Wilks’ Lambda treatment (p = 0.94), time (p = 0.001), gender (p = 0.001), treatment

time

(p = 0.11), treatment

gender (p = 0.90), time

gender (p = 0.001), and treatment

time

gender

(p = 0.57) effects. The univariate analysis revealed that although some time and gender effects were

observed, no signiﬁcant treatment

time or gender

time effects were seen in bench press 1RM, bench

press endurance, leg press 1RM, or leg press endurance. A signiﬁcant treatment

time

gender

interaction was observed in leg press endurance (p = 0.04). Post-hoc analyses revealed that leg

press endurance signiﬁcantly increased from baseline measurements in the CNL female group

after 5 days of supplementation, but these values were not different among treatments. Figure 5

presents the mean change from baseline with 95% CIs for these variables. Performing the initial

1RM test and three sets of 10 repetitions at 70% of 1RM, with the last set to failure, promoted

fatigue, as expected. Therefore, recovery bench press and leg press 1RM signiﬁcantly decreased

in all treatments. After 5 days of supplementation, 1RM bench press performance was signiﬁcantly

higher than baseline values following CNH treatment (PLA: 0.7 [

−

1.6, 3.0], CNL: 2.0 [

−

0.9, 4.9],

CNH: 6.1 [3.5, 8.7] kg, p = 0.01). Participants in the CNH treatment also tended to experience a lower

degree of loss in 1RM strength during the recovery performance tests following supplementation,

on day 5 (PLA:

−

9.3 [

−

13.5,

−

5.0], CNL:

−

9.3 [

−

13.5,

−

5.1], CNH:

−

3.9 [

−

6.6,

−

1.2] kg, p = 0.07).

After 5 days, pre-supplementation 1RM leg press was signiﬁcantly increased above baseline values,

only with CNH treatment (PLA: 13.9 [

−

15.7, 43.5], CNL: 14.6 [

−

0.5, 29.7], CNH: 24.7 [8.8, 40.6] kg,

p = 0.73) and participants were able to maintain strength during the recovery while experiencing a

signiﬁcant decrease in 1RM leg press strength from baseline values in the PLA and CNL treatments

(PLA:

−

30.5 [

−

53.4,

−

7.7], CNL:

−

29.0 [

−

49.5,

−

8.4], CNH:

−

13.3 [

−

31.9, 5.3] kg, p = 0.42). In terms

of muscular endurance, participants in the CNL treatment were able to perform more bench press

repetitions at 70% of their 1RM during recovery after supplementation on day 5 (PLA: 0.4 [−0.8, 1.6],

CNL: 0.9 [0.35, 1.5], CNH: 0.5 [

−

0.2, 0.3], p = 0.56), more leg press repetitions at 70% of 1RM prior to

supplementation on day 5 (PLA:

−

0.2 [

−

1.6, 1.2], CNL: 0.9 [0.2, 1.6], CNH: 0.2 [

−

0.5, 0.9], p = 0.25)

and after supplementation during recovery on day 5 (PLA:

−

0.03 [

−

1.2, 1.1], CNL: 1.1 [0.3, 1.9],

CNH: 0.4 [−0.4, 1.2], p = 0.23).

Nutrients 2017, 9, 1359 12 of 22

Table 2. Bench Press and Leg Press Performance.

Variable

Treatment

Day

Mean

Interaction

p-Level

0 Pre 0 Post 5 Pre 5 Post

Bench Press Overall 73.9 ± 30.0 67.9 ± 28.4 * 75.2 ± 30.4 * 70.5 ± 28.9 * 71.9 ± 29.4 Time 0.001

1RM (kg) PLA 74.4 ± 30.7 68.1 ± 28.3 74.8 ± 30.4 70.3 ± 29.2 72.5 ± 29.1 Treatment 0.94

CNL 73.1 ± 29.7 67.1 ± 28.3 74.1 ± 30.2 69.0 ± 28.7 70.4 ± 28.8 Treatment × Time 0.46

CNH 74.1 ± 30.9 68.8 ± 29.7 77.0 ± 31.6 72.4 ± 30.1 73.0 ± 30.7

Male 92.4 ± 19.9 84.4 ± 21.2 * 93.7 ± 20.5 * 87.6 ± 21.1 * 89.3 ± 21.0 Gender 0.001

Female

40.5 ± 7.7

†

38.2 ± 8.4

†,

* 41.8 ± 8.8

†,

* 39.6 ± 7.6

†

40.0 ± 8.6

†

Time × Gender 0.001

PLA M 92.8 ± 21.1 84.0 ± 21.6 92.8 ± 21.2 86.9 ± 22.1 88.9 ± 21.2

Treatment

Gender

0.95

PLA F 41.3 ± 9.4 39.3 ± 9.1 42.2 ± 9.9 40.2 ± 8.2 41.0 ± 9.3 T × T × G 0.96

CNL M 91.4 ± 19.7 83.4 ± 21.1 92.2 ± 20.6 85.8 ± 20.8 87.3 ± 20.8

CNL F 40.2 ± 6.8 37.5 ± 7.5 41.3 ± 8.6 38.6 ± 7.1 39.4 ± 7.8

CNH M 93.1 ± 20.0 85.9 ± 22.1 96.2 ± 20.8 90.2 ± 21.2 91.7 ± 21.2

CNH F 40.5 ± 7.7 37.9 ± 9.2 42.0 ± 8.9 40.2 ± 8.1 39.7 ± 8.6

Bench Press Overall 14.1 ± 5.3 14.1 ± 4.7 14.7 ± 5.0 15.5 ± 5.3 * 14.3 ± 5.1 Time 0.006

Endurance PLA 14.8 ± 5.9 14.0 ± 5.0 14.7 ± 5.6 15.6 ± 6.3 14.8 ± 5.8 Treatment 0.55

(Repetitions) CNL 12.9 ± 4.0 14.0 ± 4.4 14.1 ± 4.6 14.9 ± 4.5 13.5 ± 4.1 Treatment × Time 0.76

CNH 14.8 ± 5.6 14.2 ± 4.9 15.3 ± 4.9 15.9 ± 4.9 14.7 ± 5.2

Male 13.7 ± 4.8 13.2 ± 4.7 14.1 ± 4.9 14.5 ± 5.2 13.5 ± 4.9 Gender 0.04

Female 14.9 ± 6.0 15.6 ± 4.4 15.9 ± 5.2 17.2 ± 4.9

15.8 ± 5.3

†

Time × Gender 0.34

PLA M 13.4 ± 4.6 12.7 ± 4.7 12.9 ± 3.9 14.0 ± 5.2 13.0 ± 4.5

Treatment

Gender

0.26

PLA F 17.2 ± 7.4 16.4 ± 5.0 18.0 ± 6.8 18.6 ± 7.2 18.3 ± 6.5 T × T × G 0.62

CNL M 12.6 ± 4.4 13.3 ± 5.0 13.7 ± 5.3 14.3 ± 5.2 13.0 ± 4.7

CNL F 13.4 ± 3.3 15.1 ± 3.3 14.7 ± 3.1 16.0 ± 2.6 14.2 ± 2.7

CNH M 15.2 ± 5.3 13.5 ± 4.8 15.6 ± 5.1 15.3 ± 5.5 14.6 ± 5.3

CNH F 14.0 ± 6.3 15.4 ± 5.1 14.9 ± 4.8 17.1 ± 3.7 14.8 ± 5.0

Leg Press Overall 408 ± 123 391 ± 121 * 417 ± 124 * 397 ± 122 * 403 ± 119 Time 0.001

1RM (kg) PLA 411 ± 122 397 ± 119 417 ± 125 397 ± 117 407 ± 115 Treatment 0.66

CNL 397 ± 122 379 ± 119 404 ± 122 384 ± 120 387 ± 114 Treatment × Time 0.62

CNH 417 ± 129 399 ± 128 428 ± 127 411 ± 132 414 ± 127

Male 476 ± 96 456 ± 98 483 ± 98 464 ± 97 466 ± 94 Gender 0.001

Female 286 ± 51 276 ± 50 297 ± 55 278 ± 48

287 ± 54

†

Time × Gender 0.38

PLA M 474 ± 99 457 ± 99 479 ± 105 461 ± 92 464 ± 95

Treatment

Gender

0.88

PLA F 296 ± 59 288 ± 61 305 ± 63 282 ± 46 299 ± 58 T × T × G 0.97

CNL M 463 ± 95 443 ± 94 470 ± 95 449 ± 96 448 ± 90

CNL F 278 ± 53 262 ± 44 285 ± 53 268 ± 50 275 ± 52

CNH M 491 ± 96 467 ± 104 500 ± 96 482 ± 106 485 ± 96

CNH F 284 ± 45 276 ± 47 300 ± 52 283 ± 52 286 ± 50

Leg Press Overall 20.8 ± 7.7 20.3 ± 8.1 21.5 ± 7.7 21.8 ± 7.4 21.3 ± 7.7 Time 0.06

Endurance PLA 21.8 ± 9.3 20.5 ± 8.7 21.4 ± 8.2 21.7 ± 8.2 21.8 ± 8.5 Treatment 0.7

(Repetitions) CNL 19.0 ± 7.3 18.9 ± 7.8 21.1 ± 8.3 21.4 ± 7.2 20.2 ± 7.8 Treatment × Time 0.23

CNH 21.6 ± 5.9 21.4 ± 7.9 22.0 ± 6.7 22.4 ± 6.7 21.9 ± 6.7

Male 21.6 ± 7.2 20.6 ± 6.3 21.8 ± 6.9 22.5 ± 6.5 21.4 ± 6.9 Gender 0.35

Female 19.3 ± 8.3 19.7 ± 10.6 20.9 ± 9.0 20.6 ± 8.6 21.2 ± 9.1 Time × Gender 0.56

PLA M 21.7 ± 9.1 20.2 ± 6.1 20.8 ± 6.4 22.6 ± 7.3 21.2 ± 7.5

Treatment

Gender

0.72

PLA F 21.9 ± 10.3 21.0 ± 12.4 22.3 ± 11.1 20.1 ± 10.0 22.9 ± 10.2 T × T × G 0.04

CNL M 19.9 ± 6.5 19.9 ± 6.3 20.7 ± 7.6 21.8 ± 5.4 20.3 ± 6.6

CNL F 17.5 ± 8.7 17.0 ± 9.9 21.8 ± 9.7 * 20.7 ± 10.1 20.1 ± 9.8

CNH M 23.3 ± 5.7 21.6 ± 6.8 23.9 ± 6.4 23.2 ± 7.1 22.7 ± 6.6

CNH F 18.5 ± 5.4 21.1 ± 10.0 18.5 ± 6.0 21.1 ± 6.1 20.6 ± 6.8

Data are means

SD. The multivariate analysis revealed overall Wilks’ Lambda values for treatment (p = 0.94),

time (p = 0.001), gender (p = 0.001), treatment

time (p = 0.11), treatment

gender (p = 0.90), time

gender

(p = 0.001), and treatment

time

gender (p = 0.57). Greenhouse–Geisser univariate p-levels are presented for

each variable. PLA = placebo (0 g), CNL = creatine nitrate low dose (3 g), CNH = creatine nitrate high dose (6 g),

M = male, F = female, 1RM = one repetition maximum, and T

G = time

treatment

gender interaction.

p < 0.05 is considered signiﬁcant. Statistical notations. (*) Denote a signiﬁcant difference from baseline. (

†

) Denotes

a signiﬁcant difference from male.

Nutrients 2017, 9, 1359 13 of 22

Male 21.6 ± 7.2 20.6 ± 6.3 21.8 ± 6.9 22.5 ± 6.5 21.4 ± 6.9 Gender 0.35

Female 19.3 ± 8.3 19.7 ± 10.6 20.9 ± 9.0 20.6 ± 8.6 21.2 ± 9.1 Time × Gender 0.56

PLA M 21.7 ± 9.1 20.2 ± 6.1 20.8 ± 6.4 22.6 ± 7.3 21.2 ± 7.5 Treatment × Gender 0.72

PLA F 21.9 ± 10.3 21.0 ± 12.4 22.3 ± 11.1 20.1 ± 10.0 22.9 ± 10.2 T × T × G 0.04

CNL M 19.9 ± 6.5 19.9 ± 6.3 20.7 ± 7.6 21.8 ± 5.4 20.3 ± 6.6

CNL F 17.5 ± 8.7 17.0 ± 9.9 21.8 ± 9.7 * 20.7 ± 10.1 20.1 ± 9.8

CNH M 23.3 ± 5.7 21.6 ± 6.8 23.9 ± 6.4 23.2 ± 7.1 22.7 ± 6.6

CNH F 18.5 ± 5.4 21.1 ± 10.0 18.5 ± 6.0 21.1 ± 6.1 20.6 ± 6.8

Data are means ± SD. The multivariate analysis revealed overall Wilks’ Lambda values for treatment

(p = 0.94), time (p = 0.001), gender (p = 0.001), treatment × time (p = 0.11), treatment × gender (p = 0.90),

time × gender (p = 0.001), and treatment × time × gender (p = 0.57). Greenhouse–Geisser univariate p-

levels are presented for each variable. PLA = placebo (0 g), CNL = creatine nitrate low dose (3 g), CNH

= creatine nitrate high dose (6 g), M = male, F = female, 1RM = one repetition maximum, and T × T ×

G = time × treatment × gender interaction. p < 0.05 is considered significant. Statistical notations. (*)

Denote a significant difference from baseline. (

†

) Denotes a significant difference from male.

Nutrients 2017, 9, 1359 14 of 22

Figure 5. Bench press one repetition maximum (1 RM) (A), bench press endurance (B), leg press 1 RM

nitrate (CNL), and high dose creatine nitrate (CNH) treatments. Data are mean changes (95% CI).

Confidence intervals not crossing zero are statistically significant (p < 0.05). a Represents p < 0.05

difference from PLA. b Represents p < 0.05 diﬀerence from CNL. † Represents p > 0.05 to p < 0.05 effect.

4.3.2. Cycling Time Trial Performance

Table 3 presents 4 km cycling time trial performance data. Power output data were analyzed in

absolute (W) and relative terms (W/kg

FFM) and since results were similar the absolute values are

reported. The multivariate analysis revealed overall Wilks’ Lambda treatment (p = 0.79), time (p =

0.008), gender (p = 0.001), treatment × time (p = 0.20), treatment × gender (p = 0.85), time × gender (p =

0.22), and treatment × time × gender (p = 0.06) effects. The univariate analysis revealed significant

gender (p = 0.001) and treatment × time × gender (p = 0.02) effects for time to completion and time (p

= 0.005) and gender (p = 0.001) effects for mean power. The univariate analysis revealed a statistical

trend in performance times (treatment × time effects p = 0.068, η

= 0.066) with a significant treatment

× time × gender interaction (p = 0.02). Post-hoc analyses revealed that females in the CNL treatment

performed the time trial slower (392 ± 75 to 416 ± 114 s) compared to females in the CNH treatment,

who saw improved performance (402 ± 80 to 381 ± 85 s). These values were significantly different

than day 1 performance as well as significantly different than male times. Figure 6 presents the

change in 4 km time trial time performance (seconds) and average power output observed during the

time trials. Participants experienced significant improvements in performance times from baseline

following PLA treatment, while average power output was improved over baseline values in the PLA

and CNH treatments. Improvements in performance times were nearly identical in the CNH

treatment, but were not significant, due to a larger 95% CI range observed.

Figure 5. Cont.