1. 1
Beetroot juice supplementation and its effect on 1-mile time
trial performance
Kody Springsteen • Brad Retherford • Nick Kangas • Cassidy Tokunaga
December 10, 2014
Introductions/Purpose:
Athletes are always looking for an edge against their opponent. A new ergogenic aid that
is gaining attention is the use of beetroot juice (BRJ) to enhance performance. BRJ has a high
nitrate (NO3
-
) concentration, which is a valuable substrate involved in numerous physiological
processes such as increasing blood flow, muscle contractility, and mitochondrial respiration
(Brooks et al. 2005, Wylie et al. 2012). When BRJ is consumed, anaerobic oral bacteria reduce
NO3
-
to nitrite (NO2
-
), and NO2
-
is further broken down to nitric oxide (NO) (Brooks et al. 2005,
Jones 2014b). NO has been shown in numerous experiments to provide athletes with increased
blood flow, mitochondrial respiration, and muscle contractility (Wylie et al. 2012). Due to
increased blood flow and muscle contractility, studies have shown that power output in cycling
significantly increases and time trial performance improves approximately 2-3% (Lansley et al.
2011a). Time trial performance and time to exhaustion during walking, and moderate to severe-
intensity running have also been shown to improve due to the increases in mitochondrial
respiration and reductions in submaximal VO2 levels (Lansley et al. 2011b). These experimental
results have led to many more athletes consuming beetroot juice before they compete to increase
performance. The purpose of this experiment is to look at the effects of a single dose of beetroot
juice on time trial performances in the 1-mile run.
Methods:

2. 2
The researchers gathered six male (mean ± SD: age 23.33 ± 3.39 years; body mass 81.58
± 6.79 kg; height 1.84 ± 0.04 meters; BMI 24.05 ± 1.27; Skinfold 13.1% ± 4.62% body fat;
seated blood pressure 122.50/79.33 ± 5.54/4.13 mmHg; resting heart rate 72.83 ± 8.64 BPM) and
four female (mean ± SD: age 22.25 ± 1.26 years; body mass 55.8 ± 7.05 kg; height 1.64 ± 0.07
meters; BMI 20.83 ± 1.81; Skinfold 19.2% ± 2.56% body fat; seated blood pressure
121.25/80.50 ± 2.22/4.43 mmHg; resting heart rate 73.50 ± 6.81 BPM) moderate, or highly
active volunteers from Fort Collins, CO to perform a 1-mile time trial performance over a 6-
week research period. After explanation of the experiment protocols, possible benefits, and
associated risks the subjects gave their verbal informed consent to perform in the experiment.
The volunteers performed a blind, randomized, crossover 1-mile time trial over two
weeks with the supplementation of pure beetroot juice or a placebo (Placebo consisted of water,
lime juice, and red food coloring). The volunteers were randomly given eight ounces of placebo
or pure beetroot juice, thirty minutes prior to the test. The subjects were told how the 1-mile time
trial would be performed while the researchers gathered the subjects’ anthropometric data. The
subjects then went through a fifteen-minute warm-up protocol. The warm-up consisted of a
moderate-intensity jog, as well as hamstring, quadriceps, gastrocnemius, and gluteus stretches.
The standing hamstring, quadriceps, and gastrocnemius stretch, as well as the seated gluteus
stretch were used for each subject. Once the subjects completed the warm-up, they were
provided ten minutes to get ready for the 1-mile time trial. The 1-mile time trial consisted of
running four laps around a 400-meter track. The researchers began the time trial and recorded the
time it took to complete the 1-mile run. The subjects went through a ten-minute cool down,
consisting of walking and stretching. The following week the volunteers came back and went

3. 3
through the same protocols, but with the opposite supplementation they consumed the previous
week.
The researchers performed a statistical analysis on the results of the 1-mile time trial. The
alpha level was set at p < 0.05. Statistical significance was accepted at p < 0.05. All results are
presented with mean ± sample standard deviation, unless stated otherwise. Statistics were
calculated using excel or a calculator.
Results:
Table 1 shows the anthropometric data (MALE: mean ± SD: age 23.33 ± 3.39 years;
body mass 81.58 ± 6.79 kg; height 1.84 ± 0.04 meters; BMI 24.05 ± 1.27; Skinfold 13.1% ±
4.62% body fat; seated blood pressure 122.50/79.33 ± 5.54/4.13 mmHg; resting heart rate 72.83
± 8.64 BPM; FEMALE: mean ± SD: age 22.25 ± 1.26 years; body mass 55.8 ± 7.05 kg; height
1.64 ± 0.07 meters; BMI 20.83 ± 1.81; Skinfold 19.2% ± 2.56% body fat; seated blood pressure
121.25/80.50 ± 2.22/4.43 mmHg; resting heart rate 73.50 ± 6.81 BPM) and activity level
(low/moderate/high) of each subject in the experiment. Table 2 shows the 1-mile time trial
performances (minutes: seconds) data for each subject with the beetroot juice (BRJ) and placebo,
as well as the difference (BRJ – Placebo = Difference (minutes: seconds)) between each
performance. A negative difference indicates an improvement in time trial performance when
beetroot juice was consumed and a positive difference indicates a decline in time trial
performance when beetroot juice was consumed. Figure 1 shows a side-by-side comparison for
each subjects’ 1-mile time trial performance with BRJ and placebo. Figure 2 (r = -0.3353) shows
the time differences between trials for each subject. Figure 3 shows the 1-mile time trial
performance (in seconds) versus gender and supplementation (Male BRJ: 401.17 ± 33.25
seconds; Male w/o BRJ: 402 ± 34.42 seconds; Female BRJ: 502.25 ± 65.97 seconds; Female w/o

4. 4
BRJ: 504.5 ± 65.75 seconds). The male p-value = 0.483 and female p-value = 0.482 was much
greater than the alpha level = 0.05.
Table 1: Anthropometric Data
Table 2: 1-Mile Time Trial Data
Subject
#
Gender Age
(years)
Weight
(kg)
Height
(meters)
BMI Skinfold
(% BF)
BIA
(%
BF)
Resting
HR(bpm)
BP(mmHg)
Activity
Level
1 Female 21 50 1.63 18.8 15.7 17.4 68(124/86) Moderate
2 Male 27 79.4 1.83 23.7 14.3 13.1 62(118/80) High
3 Male 28 86.3 1.88 24.4 16.1 14.9 74(129/84) High
4 Male 21 70.3 1.80 21.7 5.2 6.8 64(121/76) High
5 Male 21 88.4 1.88 25 10.8 10.4 74(115/74) Moderate
6 Female 22 62.1 1.73 20.7 21.7 18.7 82(122/76) Moderate
7 Female 22 61.7 1.63 23.2 20.3 19.4 76(119/82) Moderate
8 Male 23 78.8 1.80 24.3 13.8 12.2 85(124/78) Moderate
9 Male 20 86.3 1.85 25.2 18.4 18.2 78(128/84) Moderate
10 Female 24 49.4 1.55 20.6 19.1 16.4 68(120/78) Moderate
Subject # Gender
Time with BRJ
(minutes: seconds)
Time Without BRJ
(minutes: seconds)
Difference
(BRJ Time- w/o BRJ Time)
1 Female 9:56 9:59 -0:03
2 Male 5:56 5:57 -0:01
3 Male 7:05 7:01 +0:04
4 Male 6:05 6:02 +0:03
5 Male 7:08 7:13 -0:05
6 Female 7:23 7:27 -0:04
7 Female 8:14 8:11 +0:03
8 Male 6:42 6:38 -0:04
9 Male 7:11 7:09 -0:02
10 Female 7:56 8:01 -0:05

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Figure 1: Subjects’ 1-mile time trial times with and without BRJ.
Figure 2: 1-mile time trial differences. (r = -0.3353)(Time difference = BRJ Time – Placebo
Time)
4:48
6:00
7:12
8:24
9:36
10:48
1
2
3
4
5
6
7
8
9
10
Run
Time
(min:sec)
Subjects
Subjects'
Run
Times
Time
with
BRJ
Time
Without
BRJ
r=
-­‐0.3353
-­‐6
-­‐5
-­‐4
-­‐3
-­‐2
-­‐1
0
1
2
3
4
5
1
2
3
4
5
6
7
8
9
10
Time
Difference
(Seconds)
Subject
#
Running
Time
Diferences
for
each
Subject

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Figure 3: 1-mile time trial performance versus gender and supplementation. Male BRJ: 401.17 ±
33.25 seconds; Male w/o BRJ: 402 ± 34.42 seconds; Female BRJ: 502.25 ± 65.97 seconds;
Female w/o BRJ: 504.5 ± 65.75 seconds. Male p-value = 0.483 and female p-value = 0.482, both
are > 0.05.
0
100
200
300
400
500
600
Male
BRJ
Male
w/o
BRJ
Female
BRJ
Female
w/o
BRJ
Time
(seconds)
Gender
and
Supplement
Average
1-­‐Mile
Time
Trial
Performance
vs
Gender
&
Supplementation
Male
BRJ
Male
w/o
BRJ
Female
BRJ
Female
w/o
BRJ

7. 7
Beetroot Juice Literature Review
Nitrate (NO3
-
) supplementation is an up and coming ergogenic aid that recent research
has shown to have enormous benefits for athletes. NO3
-
is a naturally occurring inorganic
molecule found in several dietary forms. One of the best sources of dietary NO3
-
is found in
beetroots, which is why beetroot juice (BRJ) supplementation has been a recent focus as an
ergogenic aid for athletes. The mechanism by which NO3
-
is utilized by the body is through
anaerobic oral bacteria reducing NO3
-
to nitrite (NO2
-
) (Jones 2014b). NO2
-
is further reduced to
nitric oxide (NO) and can then be utilized by the body. NO has been shown to increase
mitochondrial respiration, blood flow, muscle contractility, and calcium (Ca2+
) handling (Wylie
et al. 2012).
The current bodies of literature seem to agree that BRJ supplementation can increase
muscle endurance and efficiency during continuous, extended endurance exercise by about 15-
25% (Jones 2014a). This improvement in muscle efficiency may occur because of restricted
oxygen availability. When oxygen is restricted, similar to when exercising, the NO2
-
reduction
pathway is under higher demand to produce NO than the L-arginine pathway. By increasing the
availability of NO2
-
through BRJ supplementation, an individual has more substrates to produce
NO. Through NO’s ergogenic effects, enhanced Ca2+
handling and muscle contractility, muscle
efficiency is improved. However, it should be noted that this mechanism requires further
research to validate these findings (Jones 2014a).
Additionally, current literature substantiates the claim that BRJ supplementation can
improve time trial performance. One study found that cyclists with the supplementation of BRJ

8. 8
improved time trial performance in the 4 km and 16.1 km by 2.7%-2.8% when compared to the
placebo (Lansley et al. 2011a)(Jones 2014b). Also Murphy et al. saw improvements in 5-km
treadmill time trials for “recreationally fit” adults following the consumption of baked beetroot
(Jones 2014b). This improvement is attributed to an increase in power output, spared
intramuscular phosphocreatine (PCr) concentration, and a reduction in submaximal VO2,
allowing the athlete to sustain an increased power output at submaximal workloads for longer
periods of time (Jones 2014b). NO3
-
consumption changes muscle energy metabolism during
exercise, by lowering the ATP cost of muscle power production (Bailey et al. 2010). This is
believed to be because of NO and its enhancement of Ca2+
handling at the sarcoplasmic
reticulum and actin-myosin sites. BRJ has additionally been shown to increase NO concentration
in the muscle, thereby increasing blood flow and oxygen delivery to the muscles. This may be
another mechanism that plays a significant role in the athlete’s endurance performance and
mitochondrial respiration.
The majority of beetroot supplementation research was conducted in order to study the
effects of NO3
-
consumption on high-intensity, long duration, continuous endurance exercise. In
spite of this, Lee J. Wylie et al. studied the effect of beetroot supplementation on high-intensity,
intermittent exercise. This type of exercise is common for team sports and uses drastically
different metabolic pathways. For example, steady state, long distance running mostly uses
oxidative phosphorylation as the metabolic pathway to supply energy. In contrast, intermittent,
high-intensity anaerobic exercise uses more of the PCr and glycolytic pathways to supply energy.
Therefore, it is important to understand whether the same ergogenic effects of BRJ will be seen
during sporadic anaerobic exercise, when compared to long-distance aerobic exercise.

9. 9
The results of Lee J. Wylie’s study showed that beetroot supplementation increased
performance of subjects performing high-intensity, sporadic exercises. This was shown by an
increase of 4.2% in distance to completion during a YO-YO IR1 test with the consumption of
BRJ when compared to the control (Wylie et al. 2012). YO-YO IR1 is an intermittent interval
endurance test designed to measure exercise performance similar to a beep test. This test is
beneficial for team sports athletes because of the 5 to 10 second breaks between intervals, which
simulate a game environment. The significance of this study was that it showed that NO3
-
supplementation as an ergogenic aid is not only practical for long-distance endurance athletes,
but could potentially be practical and beneficial for recurrent anaerobic exercise, like team
sports.
One current body of literature by Marco Pinna et al., studied the effects of beetroot
supplementation on aerobic performance during swimming. This study differed from other
studies because the main areas of study for BRJ have been on aerobic performance for cycling,
walking, and running. Pinna and colleagues found that the aerobic energy cost (AEC) decreased
and the workload at anaerobic threshold increased, suggesting that beetroot supplementation
improves swimming performance. This study was important because the specificity of exercise
can have a large impact on the results of a study. Therefore, the effects of any ergogenic aid
should be tested specific to the exercise to ensure accurate results.
Although there has been an increase in the number of NO3
-
supplementation studies in
recent years, there are still several unknowns regarding its ergogenic effects. According to
current literature, the exact mechanism for which NO3
-
supplementation influences muscle
metabolism, as well as energy substrate utilization and availability still needs to be further

10. 10
researched. Current research accepts that there are two main mechanisms that utilize dietary
NO3
-
and convert it to NO. The first pathway is the use of L-arginine and oxygen being
catalyzed by nitric oxide synthase (NOS) to make NO3
-
and NO2
-
. Once these substrates are
created they can be utilized by the second pathway, NO3
-
-NO2
-
-NO pathway. This pathway uses
anaerobic oral bacteria and xanthine oxidase to reduce NO3
-
and NO2
-
to NO, so that the body
can utilize NO. Although these two mechanisms are well understood, how these mechanisms
influence muscle metabolism, energy substrate utilization, and Ca2+
handling is contentious [1,3-
7].
Clearly, NO3
-
supplementation has increased in popularity and has been studied more in
recent years. From the bodies of literature discussed, all of them conclude that BRJ
supplementation decreases the oxygen cost of exercise, improves time trial performance,
increases power output, and increases VO2 kinetics (mitochondrial respiration). However, more
research is needed on the dose-response relationship of BRJ, sport specificity, and the specific
metabolic pathways that BRJ effects during athletic performance. The focus of our study was to
see if a single dose of BRJ would enhance 1-mile time trial performance. According to our
current knowledge, there are no studies focused on the effects of a single dose of BRJ on 1-mile
time trial performance. We attempted to further the current research behind the dose-response
relationship of BRJ, as well as the affects of BRJ on time trial performance.
In summary, BRJ supplementation leads to many performance enhancements such as
increased mitochondrial respiration, improved muscle contractility, decreased oxygen cost of
exercise, and increased power output during athletics. However, BRJ supplementation is in the
early stages of research and there are many unknowns requiring further research. Additional

11. 11
studies will uncover the unknowns, but the current research on BRJ supplementation shows that
it is a promising focus for up and coming athletic supplementation.

12. 12
Beetroot juice supplementation and its effect on 1-mile time
trial performance
Kody Springsteen · Brad Retherford · Nick Kangas · Cassidy Tokunaga
December 10, 2014
Technical Abstract
Purpose: Recent research on dietary nitrate supplementation with beetroot juice (BRJ) has
shown several potential ergogenic benefits for athletes. One of the indicated benefits of BRJ
supplementation has been a 2-3% increase in time trial performance for long-distance endurance
events. The present study was designed to further examine the effects of BRJ supplementation on
time trial performance during the 1-mile run.
Methods: The design of the study was a blind, randomized, crossover 1-mile time trial. The
researchers gathered four healthy females and six healthy males to perform the 1-mile run over
two weeks. Each subject completed one trial with eight ounces of BRJ supplementation and one
trial with a nitrate-free placebo ingested 30 minutes prior.
Results: The 1-mile time trial showed that: Male BRJ: 401.17 ± 33.25 seconds; Male w/o BRJ:
402 ± 34.42 seconds; Female BRJ: 502.25 ± 65.97 seconds; Female w/o BRJ: 504.5 ± 65.75
seconds. The male p-value = 0.483 and female p-value = 0.482 was much greater than the alpha
level = 0.05.
Conclusions: The findings suggested that there was not a significant variation in time trial
performance for the subjects when consuming beetroot juice compared to the placebo. This could
have been due to several factors: The short ingestion period before the trial, the short endurance
event, subject’s state of mind, and fluctuations in dietary intake. Single dose BRJ
supplementation did not improve time trial performance, but should be further researched to gain
a better understanding of the mechanisms and dose response relationship involved.

13. 13
Beetroot juice supplementation and its effect on 1-mile time
trial performance
Kody Springsteen · Brad Retherford · Nick Kangas · Cassidy Tokunaga
December 10, 2014
Lay Abstract
Purpose: Recent studies have found beneficial effects for the supplementation of beetroot juice
(BRJ) during exercise. BRJ contains chemicals that enhance performance through improved
energy usage and increased power output. The purpose of this study was to examine the effects
of BRJ on a 1-mile run.
Methods: The subjects in the study were 10 college students who were moderately active. Each
subject performed two 1-mile runs, one run with BRJ, and the other with a controlled substance.
During the study, subjects drank BRJ 30 minutes before the run, went through a warm-up,
performed the 1-mile run, and then went through a cool down.
Results: The subjects completed the 1-mile run with and without BRJ in approximately the same
time: Male BRJ: 401.17 ± 33.25 seconds; Male w/o BRJ: 402 ± 34.42 seconds; Female BRJ:
502.25 ± 65.97 seconds; Female w/o BRJ: 504.5 ± 65.75 seconds.
Conclusion: The results of the study did not support the claim that BRJ enhances performance.
Some subjects’ 1-mile run times increased, while others decreased. The subjects did not have a
large enough difference in 1-mile run times to suggest that BRJ improved their performance.
BRJ should be further researched to better understand how it works.

14. 14
References
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Boston: McGraw-Hill.
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