Senior Research Paper

The Effect of Caffeine and Sugar on the Heart Rate of Daphnia magna
Brendan Cairney and Dennis Hitaj
Macomb Mathematics Science Technology Center
AP Biology
12A
Mr. Estapa / Mrs. Gravel / Mr. Moore
4 December 2019
The Effect of Caffeine and Sugar on the Heart Rate of Daphnia magna
The purpose of this experiment was to determine how different energy
stimulants affect the heart rate of Daphnia magna. To accomplish this, caffeine,
sugar, and a non-supplement control were each mixed with spring water to
create three different solutions. This was administered to the D. magna for five
minutes. Afterward, the crustacean was subsequently placed on a well slide,
where the number of heart contractions was recorded in a ten-second interval
under a microscope. This number was multiplied by six in order to get the beats
per minute (BPM) of the heart rate. The control, caffeine, and sugar were
compared to each other to determine significance in regards to the BPM of the
Daphnia. Descriptive analysis was conducted in order to compare these
stimulants to each other. A two-sample t test was not implemented in the
experiment’s treatments, as there was not a visually apparent difference in the
box plots. Due to this, an analysis of variance (ANOVA) statistical test was run
between the three stimulants, and a P-value of 0.1933 was found. This meant
that it could not be decisively stated whether or not the treatments induced a
significant difference in BPM for Daphnia magna, as there was a 19.3% chance
that these results could have occurred by chance alone. These results are
applicable to those who consume sugary and/or caffeinated beverages, as these
drinks may increase the risk of adverse health effects. Informing the public about
the possible problems that caffeine and sugar inherently have on a human heart
can aid them in making health-conscious decisions.

Table of Contents
Introduction………………………………………………………………....…….……..1
Review of Literature………………………….……..…...…...……………………..….5
Problem Statement…………………………...………………..…..…………..….….10
Experimental Design……………….………..…………...………………..………….11
Data and Observations………………………………...………….…………..……...16
Data Analysis and Interpretation…………………..…………..………………..…...23
Conclusion………………………....……………...……………….………………..…27
Acknowledgments…………………………………….……...…….……..……...…….32
Appendix A: ANOVA
Test………………………....…..…………………………..….33
Appendix B: Professional Contacts………………………………..………………...36
Works Cited…………………………....……....…..…………………..……...………..38
Cairney - Hitaj 1
Introduction
Popular chains, such as Starbucks Coffee, are under intense scrutiny from
the public eye due to the total amount of caffeine and sugar (two common
stimulants) contained in one cup of coffee. The volume of these stimulants may,
in fact, lead to harmful health effects (Gore; Welch). At the same time, drinking
coffee is an integral part of society and a daily habit of many. With such a large
quantity of coffee being consumed, it is imperative to ask the question: what are
the specific adverse health effects attributed to the consumption of caffeine, and
other related stimulants, on the human heart?
Figure 1. U.S. & Canadian Consumption Up 19% Over Last 10 Years. Cited from Coffee Holding
Co., Inc..“Corporate Presentation” sec.gov, Coffee Holding Co., Inc., 06 Nov. 2009,
www.sec.gov/Archives/edgar/data/1007019/000110262409000526/exh99_1.htm.
Figure 1, above, displays the consumption rates of coffee, in billions of
pounds, for the United States and Canada. As shown, coffee consumption has
risen tremendously over the years, with an additional 580 million pounds of
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coffee being consumed in 2008 between the U.S. and Canada when compared
to 1998. With North Americans drinking a substantial amount of coffee every
year, it is vital to know the true effects of caffeine, as well as other stimulants, on
the heart rate of humans.
The purpose of this research was to determine how different energy
stimulants affect the heart rate of Daphnia magna. Previous research in this field
has induced conflicting results. For example, Aman Kundu and Singh Gyanesh’s
research “Dopamine Synergizes with Caffeine to Increase the Heart Rate of
Daphnia” found an increase in the heart rate of Daphnia when administered a
solution containing caffeine (254). In contrast, research conducted by A. Bichler,
A. Swenson, and M.A. Harris entitled “A Combination of Caffeine and Taurine
Has No Effect on Short Term Memory but Induces Changes in Heart Rate and
Mean Arterial Blood Pressure” concluded that a caffeine-based solution reduced
the heart rate of humans (471-476). Therefore, the research conducted
throughout the rest of this paper seeks to clarify the true effect that caffeine and
sugar have on the heart rate of humans by using Daphnia magna as a model.
This will help the public make informed decisions that positively affect their
health.
At a molecular level, both caffeine and sugar have direct effects on the
heart. For example, when caffeine is ingested, it releases hormones that
subsequently act on the heart to release norepinephrine. This forces the heart to
contract, producing a similar effect to adrenaline. Also, the levels of calcium

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inside of the cells in the heart can increase. This induces an elevated heart rate,
as the cells responsible for contracting the heart are linked to the amount of
calcium they contain (Gluckman). Likewise, when sugar is ingested, similar heart
effects can be observed. A small molecule known as “glucose 6-phosphate
(G6P), which is a product of sugar from food, was shown to cause stress to the
heart.” This stress consequently decreases function in the muscles of the heart,
which could lead to heart failure (“Too Much Sugar”).
In this experiment, both caffeine and sugar were administered to Daphnia
magna, as well as a non-supplement control (as per the advice of Doctor John D.
Helmann at Cornell University). An analytical scale was then used to measure
the caffeine and sugar. After this, solutions were created for the crustacean,
which were composed of the stimulants mixed with spring water in test tubes.
After five minutes in the solution, the Daphnia (which were placed on a well slide)
experienced 10 seconds of light exposure before data collection. This was done
under the advice of Doctor John T. Lehman, a professor at the University of
Michigan, who noted that “the light source...will progressively warm the water.”
Therefore, in order to get constant results, it was necessary to have a control
time. After the 10 seconds of light exposure passed, the heart rate of the
Daphnia magna was measured in another ten-second interval using a
microscope. The heart rate was then multiplied by six in order to get the beats
per minute (BPM) of the heart rate of the Daphnia. This method provided an
effective data collection process, which was used later in the analysis of the
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results in order to better inform the public about the true effect of caffeine and
sugar on the human heart.
This research provides useful information on the true effects of caffeine
and sugar on the human heart. Individuals could apply the information gained
from this experiment to better develop healthy habits. This could include limiting
the amount of stimulants one takes on a given day or simply foregoing drinks
with an excess of caffeine or sugar. In an era where people are increasingly
conscious of their health, having accurate information is vital to helping them
make health-centric decisions.

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Review of Literature
Energy drinks are becoming increasingly popular in the United States,
especially among young adults. In fact, the number of young adults (aged 20 to
39) who consumed energy drinks in 2016 is 1100% greater than it was in 2003
(Mishra). But how do these beverages truly affect the human heart? One study
shows that along with increasing blood pressure, energy drinks have also been
found to have adverse effects on heart rate (“Can Energy Drinks”). For this
reason, the purpose of this research was derived to study the effects of popular
ingredients, such as caffeine and sugar, used in similar drinks in order to inform
the public on any possible health risks associated with consumption.
Figure 2. Energy Drink Consumption in the United States. Image from 2011 SAMHSA Drug
Abuse Warning Network, “Energy Drink-Related Emergency Department (ED) Visits, by Year:
2005 to 2011” The CBHSQ Report,
www.ncbi.nlm.nih.gov/books/NBK384664/figure/SR-126_RB-2460.f1/.
Figure 2, above, displays the number of American emergency department
visits resulting from energy drink consumption from 2005 to 2011. There is a
clear upward trend in this timespan, with 1,494 visits in 2005 to 20,783 visits in
2011.
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The amount of emergency room visits related to energy drinks has
increased over recent years. This elicits the question: what adverse health
effects are exhibited from these beverages? It is normal for the heart rate of an
individual to increase significantly when experiencing stress, trauma, or engaging
in physical activity. However, a high resting heart rate, or tachycardia, can induce
unconsciousness, heart attacks, and cardiac arrest, while also “compromis[ing]
blood flow to the rest of the body” (“Tachycardia: Fast Heart”).
Research has been done to deepen the understanding of the effects that
energy stimulants have on heart rate. The issue is, testing human heart rate can
be challenging and filled with lurking variables. One way to solve this problem is
through the use of Daphnia magna, a crustacean barely visible to the naked eye.
In an experiment conducted by Arturo Villegas-Navarro (from Laboratorio de
Investigación), Esperanza Rosas (from San Jose State University), and José L.
Reyes (from Centro de Investigación y de Estudios Avanzados), Daphnia magna
were administered four different pharmaceutical drugs (ouabain, verapamil,
metaproterenol, and metoprolol) in order to analyze their effect on heart rate. A
microscope, along with image processing, was utilized to observe the D. magna,
where the heart rate was measured. A one-way ANOVA and t test were used to
determine statistically significant treatments. It was found that metaproterenol
significantly increased the heart rate of Daphnia magna by an average of 5% to
20%. Metoprolol, for the most part, increased heart rate of the crustacean by a
similar margin. Finally, the four drugs exhibited toxicity to Daphnia, which would
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also be the result when compared to mammals. This experiment demonstrated
the effectiveness of using Daphnia magna as a suitable substitute for the human
heart and is the basis for the research that will be conducted.
Figure 3. Daphnia Magna. Reproduced from Ted Kinsman, “Water Flea Daphnia Magna Greeting
Card” Fine Art America,
www.fineartamerica.com/featured/8-water-flea-daphnia-magna-ted-kinsman.html?product=greetin
g-card.
Figure 3, above, displays the anatomy of a Daphnia magna specimen. As
shown, the internals are clearly visible and discernable. More specifically, the
heart is detectable through the crustacean’s translucent body. This inherently
makes the Daphnia an excellent creature for measuring changes in heart rate
caused by the administration of different quantities of caffeine or sugar.
Deciding which energy stimulants to use in this experiment was critical in
order to effectively apply this research to the daily life of people around the globe.
It was first determined that regardless of which energy stimulants were used, a
non-supplement control needed to be issued in order to eliminate any potential

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lurking variables. The Daphnia exposed to the control treatment were also the
base measurement of heart rate used to determine if different energy stimulants
had an effect on heart rate. As stated above, sugar is a vital ingredient in the
production of energy drinks (and also other beverages such as coffee). An
experiment conducted by Doctor David O. Kennedy and Doctor Andrew B.
Scholey found that test subjects who were administered sugar in the form of pills
had a higher heart rate than test subjects who were given a placebo. For these
reasons, sugar was one of the stimulants tested. It was also decided that a
caffeine supplement would be used to test the effect of caffeine on heart rate.
This selection was a straightforward choice because over 210 million Americans
drink at least one cup of coffee every morning (“Americans Are Drinking”). In a
2018 study conducted by Aman Kundu from Lovely Professional University and
Singh Gyanesh from Indian Institute of Technology Kanpur, caffeine resulted in
an increase in the overall heart rate of Daphnia magna, so a similar result is
expected in humans.
Kundu and Gyanesh’s research is one of the many experiments that have
been conducted in this field of study. Their experiment tested the effect of
dopamine, ethanol, and caffeine on the heart rate of Daphnia magna. They did
this by analyzing the heart rate of the crustacean through a computer-aided
microscope, which had real-time imaging capability. A paired t t est was
performed comparing the different treatments, and it was found that dopamine
and caffeine increase the heart rate of Daphnia magna, while ethanol decreases
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the heart rate of Daphnia. The setup of this experiment will be modeled after
Kundu and Gyanesh’s experiment, but different heart rate stimulants--caffeine
and sugar--will be used, as well as randomization being clearly stated to ensure
validity of the data found in the experiment.
Overall, understanding the effects of energy stimulants on the human
heart is essential in order to promote healthy lifestyle choices. This is why, using
previous research as a basis, the experiment conducted in this paper will find the
effects of energy stimulants on the heart rate of Daphnia magna and compare
them to the human heart.

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Problem Statement
Problem:
To determine how different energy stimulants affect the heart rate of
Daphnia magna.
Hypothesis:
The caffeine and sugar stimulants will increase the average resting heart
rate in Daphnia magna, with caffeine increasing the average resting heart rate
the most.
Data Measured:
The independent variable was the type of energy stimulant used. These
included caffeine supplements (25 milligrams), sugar (50 milligrams), and a
non-supplement used as a control. The dependent variable was the heart rate of
Daphnia magna, measured in beats per minute. Descriptive analysis and an
analysis of variance (ANOVA) statistical test were used to determine how the
different energy stimulants affect the heart rate of Daphnia magna. The research
was completed with 15 trials of caffeine supplements, 15 trials of sugar, and 15
trials of a non-supplement control.

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Experimental Design
Materials:
(45) Daphnia magna (3) Well Slides

375 mg Kaged Muscle Caffeine Spring Water
Supplements (3) Test Tubes
750 mg Anthony’s Organic Cane Sugar Test Tube Holder
(3) Pipettes Mortar and Pestle
Laptop (2) Phones (Clock & Counter Apps)
TI‑Nspire CX Handheld Weigh Boat
Graphing Calculator Funnel
25 mL Graduated Cylinder Analytical Scale 0.0001g
Specimen Dish Precision
Swift SM - 80 Microscope
Procedures:
Randomization
1. Assign a randomized treatment to the Daphnia using the randInt(1,3) function

on the TI‑Nspire CX handheld graphing calculator. 1 represents the control, 2
represents the caffeine treatment, and 3 represents the sugar treatment.
2. If the function outputs a 1, complete steps 6-15.
5. Repeat steps 1-4 until 15 trials of each treatment have been completed.
Control
6. Fill the graduated cylinder with five mL of spring water.
7. Pour the five mL of spring water into a test tube.
8. Using a pipette, extract a Daphnia magna f rom the colony and place it into the
test tube.
9. Wait five minutes for the D. magna to remain in the solution.
10. Pour the solution, along with the Daphnia, onto the specimen dish.
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11. Using the pipette, extract the Daphnia from the specimen dish and place it on
a well slide.
12. Place the well slide underneath the microscope.
13. Using the Clock App to count ten seconds, turn on the microscope light to
level two and focus the lens on the crustacean.
14. Count the number of heart contractions for the Daphnia underneath the
microscope using the Counter App. Ensure that this is done in a period of ten
seconds using the Clock App.
15. Record the data in a spreadsheet, making certain to multiply the data by six to
get the BPM of the Daphnia.
Caffeine
16. Grind one supplement of Kaged Muscle caffeine using the mortar and pestle
until the supplement has been ground into a fine powder.
17. Weigh out 25 mg of the caffeine powder on a weight boat using the analytical
scale.
18. Using the funnel, fill the test tube with the 25 mg of caffeine powder from the
weigh boat.
21. Mix the caffeine powder and spring water by gently swirling the test tube for
ten seconds to let the caffeine powder dissolve.
test tube.
a well slide.

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Sugar
30. Weigh out 50 mg of Anthony’s Organic Cane Sugar on a weight boat using
the analytical scale.
31. Using the funnel, fill the test tube with the 50 mg of sugar from the weigh
boat.
34. Mix the sugar and spring water by gently swirling the test tube for ten seconds
to let the sugar dissolve.
test tube.
a well slide.
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Diagram:
Figure 4. Experimental Setup
Figure 4, above, displays the setup used in the experiment. Note that the
well slide was placed underneath the microscope at a magnification level of 5x
for detailed viewing.

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Figure 5. Materials
Figure 5, above, displays the materials used in the experiment. Note that
only one of the three Daphnia magna colonies that were used is shown.
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Data and Observations
This section includes the data produced in the experiment, along with
several observations made throughout the trials. It includes the heart rate,
measured in beats per minute (BPM), of Daphnia magna when administered
sugar, caffeine, or a non-supplement control.
Data:
Table 1
Heart Rate of Daphnia magna in the Control Treatment
Trial Heart Rate (BPM)
1 228
4 216
7 204
10 198
12 234
14 312
15 192
19 198
20 408
21 312
22 312
24 414
26 354
28 300
31 420
Average 286
S.D. 83
Table 1, above, displays the heart rate in beats per minute of the control
trials run on Daphnia magna. The BPM was found by counting the number of
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heart contractions made by the Daphnia in a period of ten seconds and then
multiplying it by six. A total of 15 control trials were conducted. Trials 1-19 were
completed on day one of data collection, while trials 20-31 were done on day
two. The average beats per minute of the Daphnia for the control trials, along
with the standard deviation (S.D.) are also displayed in Table 1.
Table 2
Heart Rate of Daphnia magna in the Caffeine Treatment
2 216
3 138
5 198
11 258
16 186
25 438
30 384
38 360
39 264
40 396
41 414
42 258
43 420
44 426
45 402
Average 317
S.D. 103
Table 2, above, displays the heart rate in beats per minute of the caffeine
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multiplying it by six. A total of 15 caffeine trials were conducted. Trials 2-16 were
two. The average beats per minute of the Daphnia for the caffeine trials, along
with the standard deviation (S.D.) are also displayed in Table 2.
Table 3
Heart Rate of Daphnia magna in the Sugar Treatment
6 360
8 258
9 264
13 288
17 450
18 402
23 306
27 432
29 366
32 306
33 372
34 288
35 300
36 474
37 312
Average 345
S.D. 69
Table 3, above, displays the heart rate in beats per minute of the sugar
multiplying it by six. A total of 15 sugar trials were conducted. Trials 6-18 were
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two. The average beats per minute of the Daphnia for the sugar trials, along with
the standard deviation (S.D.) are also displayed in Table 3.
Observations:
Table 4
Observations Pertaining to Daphnia magna
Trial Treatment Observation
The Daphnia was extremely lethargic with a weak
3 Caffeine
heartbeat.
ied while in the sugar solution. This trial
The Daphnia d
4 Sugar
was negated.
The Daphnia r emained on the test tube while it was
5 Caffeine
being poured out into the specimen dish.
6 Sugar The Daphnia had a small body and mass.
7 Control ad green colored intestines.
The Daphnia h
8 Sugar The Daphnia had green colored intestines.
The microscope’s lightbulb was on for three additional
9 Sugar
seconds.
12 Control ad a small body and mass.
The Daphnia h
18 Sugar The Daphnia had green colored intestines.
The Daphnia had a l arge mass and green colored
19 Control
intestines; it also carried three eggs.
The Daphnia h ad a large mass. Also, the microscope’s
20 Control
lightbulb was turned on three seconds late.
This trial marks the beginning of the second day. A
substantial amount of Daphnia had died between day
21 Control
one and day two. Also, several babies were observed
in this colony.
as extremely fast. Also, two newborn
The Daphnia w
22 Control
Daphnia were inside the test tube.
29 Sugar The Daphnia had very developed eggs.
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Trial Treatment Observation

31 Control The timer was started one second late.
33 Sugar The Daphnia had a large body.
From this point forward, a new colony was used for
34 Sugar
data collection.
38 Caffeine The Daphnia’s heart was difficult to see.
39 Caffeine The Daphnia had big eggs.

The Daphnia had a large body mass. Also, the timer
42 Caffeine
started five seconds too late.
44 Caffeine The Daphnia had one egg.
Table 4, above, displays a list of observations regarding the trials of the
experiment. It should be noted that the Daphnia were in a dark fridge for the
weekend prior to data collection. This could have, consequently, resulted in
fluctuations in heart rate.
Figure 6. Measuring the Stimulants

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Figure 6, above, displays the pouring and measuring of the sugar used in
the experiment. The sugar was poured onto a weigh boat and subsequently
placed on an analytical scale, where a weight of 50 milligrams was obtained. The
same process was used for caffeine, but this stimulant was measured to 25
milligrams instead.
Figure 7. Preparing the Solution
Figure 7, above, shows the process used to prepare the solution that the
Daphnia magna absorbed. The picture on the left displays the energy stimulant
being poured into the test tube through the funnel, while the picture on the right
shows the spring water being poured into the test tube to create the solution. The
Daphnia was then submerged in the solution for five minutes.

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Figure 8. Measuring Heart Rate Through the Microscope
Figure 8 displays the process of measuring the heart rate of Daphnia.
First, a Daphnia magna was placed onto a well slide, with minimal water
surrounding it. The researcher proceeded to count the number of heart
contractions exhibited by the crustacean using the Counter application. This
number was subsequently multiplied by six (to calculate BPM) and then recorded
in a spreadsheet.
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Data Analysis and Interpretation
This experiment was conducted to determine the effect of caffeine and
sugar on the heart rate of Daphnia magna. To test this, the number of heart
contractions in a ten-second interval was tracked using the Counter app. Next,
this number was multiplied by six in order to get heart rate in BPM. An analysis of
variance (ANOVA) test was conducted in order to determine if caffeine or sugar
yielded a statistically different BPM in the Daphnia magna when compared to a
non-supplement control.
The data for each trial were compared to each other and non-supplement
control. Each trial in the experiment was held constant, as the same amount of
time was allotted for each supplement to dissolve in spring water, and the overall
time of light exposure was identical among trials. This helped ensure that similar
conditions would be applied to each of the three treatments, reducing the chance
of lurking variables. To reduce the possibility of human error, the same
researcher counted the heart rate for each trial so that the BPM in each trial was
obtained in a similar fashion. The order in which the trials were conducted was
randomly chosen, which reduced bias as each treatment had an equally likely
chance of being selected. Each stimulant was assigned a number (1-3) and
randomly selected. The experiment was conducted 15 times for all three
solutions to increase the likelihood of normal data. After 15 trials were conducted
for each treatment, no extreme skewness or outliers were observed in the
graphs. Therefore, the data collected was valid and able to be used for a
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statistical test.
Figure 9. Box Plots of Heart Rates for Different Supplements
Figure 9, above, displays distributions for the control, caffeine, and sugar
trials. The dotted lines represent the mean BPM for each box plot, and the solid
lines represent the median for each distribution. The caffeine treatment had the
most variability and was left-skewed, with the mean almost 43 BPM less than the
median. Visually, the three box plots overlap with one another. Most apparently,
the control box plot overlapped with the entire caffeine box plot, as 100% of its
data fell between the range of the caffeine box plot. As such, an ANOVA test
needed to be conducted to determine if there was a significant difference
between the treatments. This can be conducted because the data meets the
three assumptions of an ANOVA statistical test. First, there are three

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independent simple random samples (one from each of the three populations).
Second, each population has no outliers or extreme skewness. Finally, the
largest standard deviation (103.4 for caffeine) is less than two times the smallest
standard deviation (69.0 for sugar).
H0: μ control = μ caf f eine = μ sugar
Ha: Not all μ control , μ caf f eine , and μ sugar are equal.
Figure 10. Null and Alternative Hypotheses
Figure 10, above, states the null and alternative hypotheses for the
ANOVA test. The null hypothesis stipulates that the average heart rate of
Daphnia not administered a treatment will equal the average heart rate of
Daphnia exposed to caffeine, and that will equal the average heart rate of
Daphnia exposed to sugar. The alternative hypothesis states that not all of the
means are equal to each other.
Table 5
Results of ANOVA Test for Control, Caffeine, and Sugar
ANOVA Test
F 1.709391793
PVal 0.1933257155
df 2
dfError 42
SS 25593.6
MS 12796.8
Table 5, above, displays the values that were found after running an
ANOVA statistical test. The test produced a P-value of 0.1933, which is greater
than the accepted alpha level of 0.05. The null hypothesis, which states that the
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treatment means are equal to each other, failed to be rejected. This led to the
conclusion that there were no significant difference in the heart rate of Daphnia
magna between the energy stimulants tested.
Based on the box plots, both caffeine and sugar increased the overall
heart rate of the Daphnia. More precisely, the sample means of the treatments
increased to 317.2 BPM for the caffeine and 345.2 BPM for the sugar when
compared to the control heart rate of 286.8 BPM. Also, the bottom 50% of data
for caffeine and sugar was greater than the bottom 50% of data for the control.
However, the data was not proven to be statistically significant, as there was a
P-value of 0.1933. This meant that there was a 19.33% chance of attaining these
results by chance alone.

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Conclusion
The purpose of this research was to determine how different energy
stimulants affect the heart rate of Daphnia magna. It was hypothesized that the
caffeine and sugar stimulants would increase the average resting heart rate in
Daphnia magna, with caffeine increasing the average resting heart rate the most.
The hypothesis was ultimately rejected. In this experiment, both caffeine and
sugar were administered to the crustacean, as well as a non-supplement control.
The caffeine and sugar were measured to 25 mg and 50 mg, respectively, on an
analytical scale. Afterward, a stimulant was poured into a test tube with spring
water to create a solution for the Daphnia. After five minutes in the solution, the
crustacean was subsequently placed on a well slide where the number of heart
contractions was recorded in a ten-second interval. This number was multiplied
by six in order to get their beats per minute (BPM). The data from the control,
caffeine, and sugar trials were compared to each other to determine significance
in regards to the BPM of the Daphnia. The objective of this research was to verify
the true effect of caffeine and sugar on the heart rate of humans, as there have
been conflicting results on this topic.
The aforementioned hypothesis was rejected because there was
significant overlap in the box plots modeling the BPM of the Daphnia created by
the three stimulants: control, caffeine, and sugar. Most apparently, the control
box plot overlapped with the entire caffeine box plot, as 100% of its data fell
between the range of the caffeine box plot. The sample means for the treatments
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were 286.8 BPM for the control, 317.2 BPM for the caffeine, and 345.2 BPM for
the sugar. Two-sample t tests were not conducted as there was not a visually
apparent difference in the box plots. Due to this, an ANOVA statistical test was
run between the three stimulants, and a P-value of 0.1933 was found. This
meant that it could not be decisively stated whether or not the treatments induced
a significant difference in BPM for Daphnia magna, as there was a 19.33%
chance that these results would occur by chance alone.
Caffeine and sugar, at a biological level, are both stimulants. Therefore,
they increase activity in the body’s nervous system (“Stimulant: Definition”).
Based on this definition, both caffeine and sugar should theoretically yield
increases in BPM due to their inherent biological properties on mammals (“Heart
Rate”). This was precisely the case in the experiment due to caffeine and sugar
having a greater mean heart rate than the control, although the results were
ultimately deemed statistically insignificant. In other words, there is a possibility,
even though it cannot be conclusively stated from this experiment, that both
caffeine and sugar increase human heart rate. At a scientific level, caffeine and
sugar ingestion have been proven to increase the level of epinephrine in a
human body, which subsequently increases heart rate (Van Soeren 999-1005;
Osborne). The results are an indicator of how caffeine and sugar affect the
human heart, which could benefit the scientific community as the results and
experimental design can be used as a baseline for future research in this area of
study.
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While not all sources agree with each other, the results of the experiment
ultimately agreed with current work done in this field by Aman Kundu, Singh
Gyanesh, D.O. Kennedy, and A.B. Scholey. For example, in Kundu and
Gyanesh’s research “Dopamine synergizes with caffeine to increase the heart
rate of Daphnia” , it was found that caffeine ultimately “increase[d] the heart rate
of Daphnia” (1-17). Also, in D.O. Kennedy and A.B. Scholey research “Glucose
administration, heart rate and cognitive performance: effects of increasing mental
effort”, sugar increased the heart rate in humans while performing cognitive tasks
(63-71). These two experiments clearly show that both sugar and caffeine
increase the heart rate in mammals. Although one of the experiments was based
on humans and the other was based on D. magna, it has been found that
Daphnia exhibit many biological similarities to mammals such as humans.
Consequently, the results of research done with Daphnia magna are applicable
to humans. These results agree with this research, as both caffeine and sugar
increased the overall heart rate when compared to the control. It should be
noted, however, that the results obtained in this experiment were statistically
insignificant, meaning that there was a relatively large chance these results
happened by chance alone.
Many errors occurred throughout this experiment that had a negative
effect on the collection of data. For example, the use of a timer was predicated
on human reaction time. If the timer was not started promptly or stopped at the
ten-second mark, there would be a slight misrepresentation in the data intertrial.

Cairney - Hitaj 30
In addition to this, it was difficult to accurately count the heart rate of the Daphnia
magna, especially at greater BPMs, due to their innately fast heart rates. Once
again, this gives a slight misrepresentation of their actual heart rate. Finally, the
quantities of caffeine (25 mg) and sugar (50 mg) used in this experiment were
not an accurate portrayal of the proportion of these stimulants used in common
beverages, which affects the real-world application of the data.
Further research should be conducted on the effect of stimulants on the
heart rate of humans. A broader range of stimulants, including guarana, green
tea extract, and paracetamol, could be tested to determine their effects on heart
rate. In addition to this, using the correct proportions of these stimulants that
correlate with real-world beverages and medication is necessary in order to gain
applicable results. Also, using a machine to promptly start and stop the timer
eliminates the reliance on human reaction time. Finally, a slow-motion camera
could be mounted to the microscope to record the heart rate of the Daphnia
magna. This would ensure the accurate collection of data. Further work in this
area would provide plentiful insight into the effect of these stimulants on the
human heart.
In conclusion, the overconsumption of sugary and caffeinated beverages
is a prevalent issue in modern-day society. Therefore, it is of the utmost
importance to find the true effect that these stimulants have on heart rate. It is
nearly impossible to make health-conscious decisions if the correct information is
not readily available and accurate. While this experiment did not produce any
Cairney - Hitaj 31
significant results, it did inform the public about the possible problems that
caffeine and sugar inherently have on a human heart. Overall, caffeine and sugar
have resulted in serious symptoms including nausea, insomnia, fatigue,
headaches, and, in some cases, death (“Diabetic Coma”; Kallmyer). By utilizing
the knowledge gained from this topic, many health-related issues could be
prevented. Whether it is cutting out the morning energy drink or simply drinking
less, incorporating better daily habits is a vital aspect of living a healthy lifestyle.
Cairney - Hitaj 32
Acknowledgments
We would like to acknowledge several individuals that aided us with the
experimental process and the research paper. First and foremost, we would like
to thank Mrs. Gravel for her continuous support in the formatting and grammar of
our paper. For aiding us tremendously in the comprehension of the science in the
experiment, as well as the overall research setup, we would like to thank Mr.
Estapa. For helping us with the statistics of the research, we would like to thank
Mrs. Cybulski. Finally, we would like to thank Professor John D. Helmann, Ph.D
from Cornell University and Professor John T. Lehman, Ph.D from the University
of Michigan for helping us understand the setup of this experiment, as well as
informing us of important details relevant to the experiment.

Cairney - Hitaj 33
Appendix A: ANOVA Test
This section of the paper gives sample calculations used to find the values
produced from the ANOVA statistical test. Note that the three populations
compared in this test were control, caffeine, and sugar.
Table 6
ANOVA Test Parameters
Population n x̄ S x̄
Control 15 286.8 83.7

Caffeine 15 317.2 103.4
Sugar 15 345.2 69
Table 6, above, displays the parameters for the three populations used in
the experiment. Each of the populations had a sample size of 15, with sugar
having the greatest mean and caffeine having the greatest standard deviation.
n 1 x 1 +n 2 x 2 +n 3 x 3
X̄ =
N
15(286.8)+15(317.2)+15(345.2)
X̄ =
45
X̄ = 316.4 BPM
̄ Calculation
Figure 11. X
Figure 11, above, displays the steps used to determine the X̄ of the three
populations. This value was later used in the calculation of the mean square
group (MSG) value.
n 1 (x 1 −x) 2 + n 2 (x 2 −x) 2 +n 3 (x 3 −x) 2

MSG =
I −1
Cairney - Hitaj 34
15(286.8−316.4) 2 +15(317.2−316.4) 2 +15(345.2−316.4) 2

MSG =
3−1
MSG = 12796.8
Figure 12. Mean Square Group Calculation
Figure 12, above, displays the steps used to determine the MSG value of
the three populations. This value was later used in the calculation of the
F-statistic.
2
(n 1 −1)S 1 + (n 2 −1)S 2 2 +(n 3 −1)S 3
2
(x 3 −x) 2
MSE =
N −I
(15−1)83.7 2 +(15−1)103.4 2 +(15−1)69 2

MSE =
45−3
MSE = 7486.1
Figure 13. Mean Square Error Calculation
Figure 13, above, displays the steps used to determine the MSE value of
the three populations. This value was later used in the calculation of the
F-statistic.
M SG
F=
M SE
12796.8
F=
7486.1
F = 1.709
Figure 14. F-statistic Calculation

Cairney - Hitaj 35
Figure 14, above, displays the F-statistic of the three populations of
caffeine, sugar, and control. This value will be used to determine the P-value of
the experiment.
I−1
df = N −I
3−1
df = 45−3
2
df = 42
Figure 15. Degrees of Freedom Calculation
Figure 15, above, displays the calculations used to determine the degrees
of freedom. This was later used with the F-statistic to deduce the P-value.
Cairney - Hitaj 36
Appendix B: Professional Contacts
This section of the paper showcases the email conversations with the
professional contacts cited in this paper. The conversation with Professor
Helmann can be found in Figure 16, and the conversation with Professor Lehman
can be found in Figure 17.
Figure 16. Professional Research Inquiry to Professor John D. Helmann Ph.D
Figure 16, above, displays the email with Professor John D. Helmann. He
is a professor at Cornell University and holds a Ph.D in the field of biology.
Professor Helmann advised the researchers to “include controls” in the
experiment. This was implemented through the use of a non-supplement control.

Cairney - Hitaj 37
Figure 17. Professional Research Inquiry to Professor John T. Lehman Ph.D
Figure 17, above, displays the email with Professor John T. Lehman. He is
a professor at the University of Michigan and holds a Ph.D in the field of zoology.
Professor Lehman helped the researchers understand the overall experimental
setup, as well as other important details, such as the importance of maintaining a
constant temperature for the solutions. For this reason, the light from the
microscope was regulated to a controlled time of 10 seconds per trial throughout
the duration of the experiment.

Cairney - Hitaj 38
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Survey.” Reuters, Thomson Reuters, 17 Mar. 2018,
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1GT0KU.
Bichler, A., Swenson, A., & Harris, M. A. (2006). A Combination of Caffeine and
Taurine Has No Effect on Short Term Memory but Induces Changes in
Heart Rate and Mean Arterial Blood Pressure. Amino Acids, 31(4),
471–476. doi:10.1007/s00726-005-0302-x
Cairney, Brendan, & Hitaj, Dennis. “Professional Research Inquiry.” Received by
Doctor John D. Helmann, 14 Sep. 2019.
Cairney, Brendan, & Hitaj, Dennis. “Professional Research Inquiry.” Received by
Doctor John T. Lehman, 17 Sep. 2019.
“Can Energy Drinks Harm Your Heart?” Cleveland HeartLab, Inc., 13 Dec. 2018,
www.clevelandheartlab.com/blog/can-energy-drinks-harm-your-heart/.
“Diabetic Coma.” Mayo Clinic, Mayo Foundation for Medical Education and
Research, 22 Aug. 2018,
www.mayoclinic.org/diseases-conditions/diabetic-coma/symptoms-causes
/syc-20371475.
Gluckman, Ty. “Ask an Expert: How Does Caffeine Affect the Heart?” Providence
Health & Services, July 2010,

Cairney - Hitaj 39
www.oregon.providence.org/forms-and-information/a/ask-an-expert-how-d
oes-caffeine-affect-the-heart/.
Gore, Alex. “How Much Caffeine Is Too Much Caffeine? The Starbucks Coffee
with Three Quarters of the Safe Daily Dose in One Cup.” Daily Mail
Online, Associated Newspapers, 7 Feb. 2013,
www.dailymail.co.uk/news/article-2274679/How-caffeine-caffeine-The-Star
bucks-coffee-quarters-safe-daily-dose-cup.html.
“Heart Rate.” Heart Rate | UC Davis Sports Medicine, University of California,
Davis, 2014,
health.ucdavis.edu/sportsmedicine/resources/heart_rate_description.html.
Kallmyer, Ted. “Caffeine Overdose Symptoms: Signs, Cases, Prevention.”
Caffeine Informer, 5 Nov. 2018,
www.caffeineinformer.com/caffeine-overdose-facts-and-fiction.
Kennedy, D. O., & Scholey, A. B. (2000). “Glucose Administration, Heart Rate
and Cognitive Performance: Effects of Increasing Mental Effort.”
Psychopharmacology, 149(1), 63 -71. doi:10.1007/s002139900335
Kundu, Aman, & Gyanesh, Singh. (2018). “Dopamine Synergizes with Caffeine to
Increase the Heart Rate of Daphnia.” F1000Research vol. 7 254. 1 Mar.
2018, doi:10.12688/f1000research.12180.1
Mishra, Manas. “Energy Drink Consumption Rising in the U.S.” Reuters,
Thomson Reuters, 27 May 2019,

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www.reuters.com/article/us-health-energy-drinks/energy-drink-consumptio
n-rising-in-the-us-idUSKCN1SX1FE.
Osborne, Dr. Wayne. “What Happens to Your Body an Hour after a Sugar
Binge.” The Independent, Independent Digital News and Media, 20 Oct.
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www.independent.co.uk/life-style/health-and-families/features/what-happe
ns-to-your-body-an-hour-after-a-sugar-binge-a6701051.html.
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www.heart.org/en/health-topics/arrhythmia/about-arrhythmia/tachycardia--f
ast-heart-rate.
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The Effect of Caffeine and Sugar on the Heart Rate of ​Daphnia magna

Brendan Cairney and Dennis Hitaj

Macomb Mathematics Science Technology Center

Mr. Estapa / Mrs. Gravel / Mr. Moore

The purpose of this experiment was to determine how different energy

minutes. Afterward, the crustacean was subsequently placed on a well slide,

where the number of heart contractions was recorded in a ten-second interval

compared to each other to determine significance in regards to the BPM of the

Daphnia​. Descriptive analysis was conducted in order to compare these

experiment’s treatments, as there was not a visually apparent difference in the

applicable to those who consume sugary and/or caffeinated beverages, as these

can aid them in making health-conscious decisions.

Data and Observations………………………………...………….…………..……...16

Data Analysis and Interpretation…………………..…………..………………..…...23

Appendix B: Professional Contacts………………………………..………………...36

other related stimulants, on the human heart?

Figure 1, above, displays the consumption rates of coffee, in billions of

to 1998. With North Americans drinking a substantial amount of coffee every

the heart rate of humans.

The purpose of this research was to determine how different energy

research “Dopamine Synergizes with Caffeine to Increase the Heart Rate of

Daphnia”​ found an increase in the heart rate of ​Daphnia​ when administered a

solution containing caffeine (254). In contrast, research conducted by A. Bichler,

A. Swenson, and M.A. Harris entitled “A Combination of Caffeine and Taurine

Mean Arterial Blood Pressure” concluded that a caffeine-based solution reduced

the heart rate of humans (471-476). Therefore, the research conducted

heart. For example, when caffeine is ingested, it releases hormones that

contract, producing a similar effect to adrenaline. Also, the levels of calcium

effects can be observed. A small molecule known as “glucose 6-phosphate

which could lead to heart failure (“​Too Much Sugar”).

In this experiment, both caffeine and sugar were administered to ​Daphnia

Helmann at Cornell University). An analytical scale was then used to measure

under the advice of Doctor John T. Lehman, a professor at the University of

Therefore, in order to get constant results, it was necessary to have a control

Daphnia magna​ was measured in another ten-second interval using a

sugar on the human heart.

This research provides useful information on the true effects of caffeine

with an excess of caffeine or sugar. In an era where people are increasingly

conscious of their health, having accurate information is vital to helping them

make health-centric decisions.

Energy drinks are becoming increasingly popular in the United States,

the public on any possible health risks associated with consumption.

Figure 2, above, displays the number of American emergency department

The amount of emergency room visits related to energy drinks has

individual to increase significantly when experiencing stress, trauma, or engaging

unconsciousness, heart attacks, and cardiac arrest, while also “compromis[ing]

blood flow to the rest of the body” (“Tachycardia: Fast Heart”).

In an experiment conducted by Arturo Villegas-Navarro (from Laboratorio de

Reyes (from Centro de Investigación y de Estudios Avanzados), ​Daphnia magna

were administered four different pharmaceutical drugs (ouabain, verapamil,

metaproterenol, and metoprolol) in order to analyze their effect on heart rate. A

determine statistically significant treatments. It was found that metaproterenol

significantly increased the heart rate of ​Daphnia magna​ by an average of 5% to

also be the result when compared to mammals. This experiment demonstrated

Figure 3, above, displays the anatomy of a ​Daphnia magna​ specimen. As

heart is detectable through the crustacean’s translucent body. This inherently

caused by the administration of different quantities of caffeine or sugar.

Deciding which energy stimulants to use in this experiment was critical in

non-supplement control needed to be issued in order to eliminate any potential

base measurement of heart rate used to determine if different energy stimulants

production of energy drinks (and also other beverages such as coffee). An

experiment conducted by Doctor David O. Kennedy and Doctor Andrew B.

Singh Gyanesh from Indian Institute of Technology Kanpur, caffeine resulted in

The Effect of Caffeine and Sugar on the Heart Rate of Daphnia magna

Daphnia. Descriptive analysis was conducted in order to compare these

Daphnia” found an increase in the heart rate of Daphnia when administered a

which could lead to heart failure (“Too Much Sugar”).

In this experiment, both caffeine and sugar were administered to Daphnia

Daphnia magna was measured in another ten-second interval using a

Reyes (from Centro de Investigación y de Estudios Avanzados), Daphnia magna

significantly increased the heart rate of Daphnia magna by an average of 5% to

Figure 3, above, displays the anatomy of a Daphnia magna specimen. As

an increase in the overall heart rate of Daphnia magna, so a similar result is

microscope, which had real-time imaging capability. A paired t t est was

Daphnia magna, measured in beats per minute. Descriptive analysis and an

(45) Daphnia magna (3) Well Slides

1. Assign a randomized treatment to the Daphnia using the randInt(1,3) function

measured in beats per minute (BPM), of Daphnia magna when administered

39 Caffeine The Daphnia had big eggs.

44 Caffeine The Daphnia had one egg.

Daphnia was then submerged in the solution for five minutes.

Figure 8 displays the process of measuring the heart rate of Daphnia.

yielded a statistically different BPM in the Daphnia magna when compared to a

H0: μ control = μ caf f eine = μ sugar

magna between the energy stimulants tested.