The Effects of Caffeine on Testosterone Levels During Aerobic Exercise
Dai Han and Chad Idris
Department of Biological Science (italic)
Saddleback College
Mission Viejo, CA 92692
As the physical fitness of an individual improves, the individual’s body secretes more
testosterone. In athletics, caffeine is known to improve an individual’s sprint, endurance, and
reduce fatigue. The question is will whether testosterone levels will be affected after aerobic
exercise while under the influence of caffeine. In this experiment, the investigators predicted
testosterone levels will be greater after aerobic exercise with caffeine than without caffeine
(Hypothesis). Adult human males (How many?) were used participate in this experiment and each
ran on a treadmill one day after taking a caffeine supplement, and ran on another day without the
caffeine supplement. Saliva samples were collected from each male before and after running with
and without caffeine. The samples were then used to measure the differences of before and after
testosterone levels for before and after aerobic exercise with and without caffeine. The mean
changes of testosterone levels for (“before and after”; or only “after”) aerobic exercise without
and with caffeine was 20.75 ± 23.74% (±SEM, N=8), and 24.15 ± 16.47% (±SEM, N=8). Analysis
of a one-tailed paired t-test for the change in testosterone before and after exercise indicated no
significant difference between aerobic exercise with or without caffeine (p = 4.5x10-1). Hence, the
data did not support the original assumption that testosterone levels will be greater with caffeine.
Introduction
Human growth hormones, such as testosterone, play a large role in muscle hypertrophy
and strength gained resulting from exercise. As the physical fitness of an individual improves,
the individual’s body secretes more testosterone. Chronic exercise is correlated with higher
productions of testosterone (Young et al., 1976). Increases in testosterone levels are attributed to
the hypertrophy and strength gained in men (Marks et al., 2000). However, a factor such as diet
will affect testosterone levels in an individual. People who eat well and eat less meat have higher
testosterone levels (Allen et al., 2002). Moreover, certain substances may enhance or decrease
testosterone levels, such as, caffeine (Beavenet al., 2008). (Should start a new paragraph)
Caffeine, a stimulant, is usually consumed by humans in infusions extracted from the
seeds of the coffee plant and the leaves of the tea brush. In athletics, moderate doses of caffeine
can improve sprint, endurance, and reduce fatigue. In contrast, too much caffeine can impair
athletic performance by interfering with coordination. Other factors include smoking and
drinking can also affect the production of testosterone (add more to make complete sentence- as
highlighted examples). People who smoke and drink will ultimately have lower testosterone
levels than those who do not smoke and drink. In addition, people who exercise regularly will
have higher testosterone levels than those who do not (Wilson et al., 1999).
Overall, the question is: Will testosterone levels be affected after aerobic exercise by
caffeine while under the influence of caffeine. Hughes et al., (1996) It is concluded that doing
aerobic exercise for twenty minutes increased testosterone secretion by twenty percent (Hughes
et al., 1996)(citation format). In this experiment, the investigators predicted testosterone levels
would be greater after aerobic exercise with caffeine than aerobic exercise without caffeine. The
results may help track-athletes, marathon-runners, and cross-fit trainers devise a caffeine
regiment in order to produce the most testosterone.
Materials and methods
Eight adult human male subjects between the ages of 18 to 26 were used participated in
this study. Subjects were residents of Orange County, CA California area (If researchers can
provide more detail about subjects, it would be great. For example: Are they athletes or nonathletes? Do they have the same frequencies in doing aerobic exercises? (3 hours/day or 30
minutes/day). All saliva samples were collected between within November 7-18, 2013. Subjects
were approached randomly. informed that the aerobic exercise will consist of running on a
treadmill, and then asked to participate in the study
Participants were asked to run on a treadmill. After agreeing to the terms of the study and
signing a consent form (if they sign, that means they agree to the terms), each subject performed
aerobic exercise without caffeine one day, and aerobic exercise with caffeine another day,
between 9 and 10 am. Subjects did not eat sixty minutes prior to data collection and were
permitted to drink water throughout the experiment. In addition, subjects did not allow ingesting
any alcohol and or caffeine products, or smoking twelve hours before testing. Moreover, subjects
rinsed their mouths thoroughly with water five minutes prior to data collection to rid their
mouths of acidic or high sugar food particles.
Subjects passively drooled into a sterile collection vial until approximately 2 mL was
collected. And after 30 minutes of running at 75% max heart rate, subjects were allowed 5
minutes to rest before collecting saliva samples. The maximum heart rate was determined by age,
following the formula:
Max heart rate = 220 - age
To maintain a constant heart rate subjects wore a Polar E600 heart rate monitor. The
procedure for aerobic exercise with caffeine was identical to aerobic exercise without caffeine,
except that subjects were given a CVS brand 200 mg caffeine supplement after a saliva sample
was collected before running, and waited 10 minutes to let caffeine come into effect. Saliva
samples were collected before and after running. Subjects ran at 75% of their max heart rate
(already mention above). The maximum heart rate was determined by age (220-age= max heart
rate). To maintain a constant heart rate subjects wore a Polar E600 heart rate monitor (should
explain before, after researcher introduced “running at 75% max heart rate”). After obtained,
samples of saliva were immediately chilled in an ice bath and transferred to a freezer within four
hours of collection for preservation until they were used in a Salimetrics™ Salivary Testosterone
EIA Kit to measure the testosterone levels in each sample before and after aerobic and anaerobic
exercise with and without caffeine. Data were recorded by using a Salimetrics™ Salivary
Testosterone EIA Kit (Researchers can go more details about the method of using EIA kit to
measure the testosterone levels in saliva samples. For example, do the saliva samples need to be
prepared by adding any solutions or stains or something else? Or those samples could be put
directly from freezer to the Salimetrics machine to generate...?)
All data were transferred to Microsoft Excel (Microsoft Corporation, Redmond,
Washington) where a four-parameter sigmoid plot (Youssef Azzazy) was generated and used to
calculate the differences of testosterone levels during aerobic exercise with and without caffeine.
Figure 2 1 shows the graph used to determine the testosterone levels in pg/mL.
1
Ratio of optical density (B/Bo)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
10
100
1000
Log of Concentration (pg/ml)
Figure 1. Ratio of optical density was plotted against log of concentration and fitted into a 4parameter sigmoid minus curve fit to determine testosterone levels. Testosterone levels were
converted by using:
Results
Analysis of a one-tailed paired t-test for the change in testosterone before and after
exercise indicated no significant difference between aerobic exercise with or without caffeine (p
= 4.5x10-1). The mean change of testosterone levels for aerobic exercise without caffeine was
20.75 ± 23.74% (±SEM, N=8). The mean change of testosterone levels for aerobic exercise with
caffeine was 24.15 ± 16.47% (±SEM, N=8). (How can researchers convert the testosterone levels
(pg/ml) to %?)
In both groups there was an increase in mean testosterone levels. Figure 1 2 shows the
mean percent change of testosterone during aerobic exercise with and without caffeine
(Researchers mentioned about figure 2? Figure one showed the ration of optical density (B/Bo)
and log of concentration (pg/ml), not the mean percent change). The graph shows overall mean
percent change in a positive direction (what does it mean? What is positive direction? Both
groups of subjects showed increase in testosterone levels? Researchers can explain more about
this), but it is important to note that individually, only 3 out of the 8 subjects with no caffeine
had an increase in testosterone levels, while compared to the 5 out of 8 subjects with caffeine
that showed an increase in testosterone levels.
The mean testosterone levels for aerobic exercise without caffeine was 40.62 pg/mL 
3.39 pg/mL (SEM, N=8) before exercise and 52.34 pg/mL  15.73 pg/mL (SEM, N=8). For
aerobic exercise with caffeine, the mean testosterone levels were 43.72 pg/mL  6.49 pg/mL
(SEM, N=8) before exercise and 52.92 pg/mL  9.40 pg/mL (SEM, N=8) after exercise. (if
researchers can create a table with those information, it will be easier to follow because what
researchers want to compare was the MEAN CHANGE of Testosterone levels before and after
aerobic exercise with and without caffeine, which were mentioned at the beginning of the results.
These highlighted numbers were considered as data summary, therefore, it is better to use a
table)
Mean Percent Change of Testosterone after
exercise (%)
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Exercise with No
Caffeine
Exercise with
Caffeine
Figure 2. Mean percent change of testosterone (%). The mean percent of testosterone for
aerobic exercise with caffeine has a larger change of testosterone than the mean percent change
of testosterone for aerobic exercise without exercise caffeine. The changes in testosterone levels
between aerobic exercise with and without caffeine did not show a There were not significant
difference in change of testosterone levels before and after aerobic exercise with and without
caffeine (one-tailed unpaired t-test, p = 0.45, N=8). Error bars are mean ±SEM.
Discussion
Subjects in this study were expected to observe a higher increase in testosterone levels
after anaerobic exercise with caffeine. Although the data showed increased levels of testosterone
for both caffeine and non-caffeine groups, there was no statistical difference between the mean
change of testosterone levels during aerobic exercise with or without caffeine (p=0.45, paired
one-tailed paired t-test, N = 8). This is not supportive of earlier studies where caffeine had
increased testosterone levels during exercise (Beaven Et al. 2008). This may have been a result
from experimental design and the exercises used for the study may have been too intense and had
unforeseen side effects. Exercise in moderation may increase testosterone levels, but in cases of
extreme workouts testosterone levels may drop. The sample size was only 8 and may not be
representative of the larger population.
The mean testosterone level for after aerobic exercise with caffeine samples were higher
than that of the mean testosterone level for aerobic without caffeine samples. Testosterone levels
fluctuate with the circadian cycle. Testosterone levels are elevated during times of sleep, and a
lack of sleep lowers testosterone levels (Luboshitzky et al, 2001). What is not yet known is that
if lack of sleep can affect testosterone production while awake. Future studies may want to
address testosterone production when subject are awake and only allowed specific amounts of
sleep.
Literature Cited
Allen, Naomi, Paul N. Appleby, Gwyneth K Davey, and Timothy J. Key. 2002. Lifestyle and
Nutritional
Determinants of Bioavailable Androgens and Related Hormones in British Men.
Cancer Causes &
Control. 13(4): p 353-363.
(Space between 2 citations)
Fahey, T.D., R.Rolph, P. Moungmee, J.Nagel and S.Morata. 1976. Serum testosterone, body
Composition and
Strength of Young Adults. Med.Sci.Sport Exerc. 8: p 31-34.
Hughes, R., J., Johnson, G., O., Housh, T., J., Weir, J., P., and Kinder, J., E. 1996. The
of Submaximal
Treadmill Running on Serum Testosterone Levels. Journal
Strength and Conditioning Research. 10(4): p 224-227.
Effect
of
Luboshitzky, R., Zabari, Z., Shen-Orr, Z., Herer, P. 2000. Disruption of the Nocturnal
Testosterone Rhythm by
Sleep Fragmentation in Normal Men. J Clin Endocrinol Metab.
86(3):1134-9. (Alphabet order) (1134-9: is it number of pages? From page 9 – 1134, or
researchers only used 1 page 1134 ? )
Wilson, Vickie, James B. McLachlan, J. Greg Falls, and Gerald A. LeBlanc. 1999. Alteration in
Sexually
Dimorphic Testosterone Biotransformation Profiles as a Biomarker of Chemically
Induced Androgen
Disruption in Mice. 107(5): p 377- 384.
Young, R., J., Ishmail, A., H., Bradley, A., and Corrigan, D., L. 1976. Effect of
Exercise of Serum
Testosterone Levels in Adult Men. 10(4): p 230-235.
Prolonged
Luboshitzky, R., Zabari, Z., Shen-Orr, Z., Herer, P. 2000. Disruption of the Nocturnal
Testosterone Rhythm by
Sleep Fragmentation in Normal Men. J Clin Endocrinol Metab.
86(3):1134-9.
Review Form
Department of Biological Sciences
Saddleback College, Mission Viejo, CA 92692
Author (s): Dai Han and Chad Idris
Title: The Effects of Caffeine on Testosterone Levels During Aerobic Exercise
Summary
Summarize the paper succinctly and dispassionately. Do not criticize here, just show that
you understood the paper.
The investigators did the research and experiment on the difference in Testosterone levels
for groups of eight adult males after doing aerobic exercise with and without caffeine. Those
subjects were asked to run on a treadmill during 9 days, from 9 – 10 AM everyday. The
participants would take 200 mg caffeine supplement (CVS brand) in one day and would not take
this supplement for another day for 9 days. The saliva samples of each subject were collected,
measured and calculated for the Testosterone levels.
Based on the results, data analysis and running one tailed paired t-test, researchers
suggested that there were not enough evidences to support the hypothesis. That meant the
Testosterone levels would not change after aerobic exercise, with or without caffeine. However,
researchers also compared their data with previous researches, which showed different results.
Researchers also gave some explanations about this issue in their paper.
General Comments
Generally explain the paper’s strengths and weaknesses and whether they are serious, or
important to our current state of knowledge.
This project is interesting and able to capture my attentions. In addition, this paper is
organized. All sections are separated well, clear and easy to follow. Researchers have a good
idea in choosing this project. It is helpful to know how caffeine, popular substances that people
use nowadays, affects the testosterone production.
However, the paper also contained some problems that need to be revised before
submitting:
- Abstract:


Methods should be a little bit more detailed.
Vocabulary used and sentences:
+ If the subjects are human, it is better to use “join” or “participate” instead of “use”.
+ Question: In the hypothesis, testosterone levels will be greater AFTER aerobic
exercise with caffeine than without caffeine. When I read the paper, researchers collected
samples, data BEFORE AND AFTER the subjects did aerobic exercises. I want to make sure
that the numbers researchers used to run data analysis and one-tailed paired t-test is both
testosterone levels BEFORE and AFTER the exercise or only AFTER exercise?
-
-
-
Introduction
 Writing in a long and “non-stopped” paragraph: the paper will be neater and easily to
follow if a long paragraph is divided into shorter ones.
 Author citation format in the body of paragraph
Methods and Materials:
 Method should be more detailed
 Arrange information to a scientific order: which one needs to be defined first? Make
sure the information can flow smoothly and make sense to readers.
Results:
Question: As I mentioned in above question, I am not sure if researchers used both data of
testosterone levels that collected BEFORE and AFTER exercises or only used data collected
AFTER exercise to calculate the mean change in percentage? If researcher can explain more
about how to convert the collected data (pg/ml) to percentage (%), results will be easier to
understand.
-
Discussion:
Explain the difference of this project with previous researches; know the reasons for the
variation. These are good points.
-
Literature Cited

Alphabet order

Double check on the number of pages of the materials that researchers used.
Technical Criticism
Review technical issues, organization and clarity. Provide a table of typographical errors,
grammatical errors, and minor textual problems. It's not the reviewer's job to copy. Edit the
paper, mark the manuscript.
This paper was a final version
x
This paper was a rough draft
There were some mistakes in using words and sentences that recommended
revising (corrections and recommendations in red were also proposed in the attached
paper).
Author citation format in the paragraph (introduction)
Alphabet order in literature cited
Overall, the paper had no important technical issues.
Recommendation
 This paper should be published as is
x
This paper should be published with revision
 This paper should not be published