This study investigated the effects of creatine monohydrate supplementation and exercise on rats. Two groups of rats were used, an experimental group given creatine monohydrate and a control group given none. The rats were tested on their performance in a running wheel, with revolutions recorded over time. The results showed that the creatine group had more revolutions on the first day, but there was already a significant pre-existing difference between the groups. Over the subsequent test days, the creatine group's performance decreased while the control group increased, suggesting creatine did not significantly increase athletic ability in rats exercising in a running wheel.

1. Running head: Effects of Creatine Monohydrate and Exercise in Rats 1
Effects of Creatine Monohydrate and Exercise in Rats
Mark Izbrand
Birmingham-Southern College
Author Note
Mark Izbrand, Psychology Department,
Birmingham-Southern College
Correspondence concerning this article should be addressed to
900 Arkadelphia Road, Birmingham, AL, 35254
maizbran@bsc.edu

2. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 2
Abstract
This study investigated whether or not Creatine Monohydrate supplementation increased athletic
performance in rats in a running wheel. We used two groups of rats, one experimental group
given Creatine Monohydrate and one control group given none. We found that Creatine
Monohydrate supplementation did not have a significant effect on the experimental group and
did not significantly increase their athletic ability. The results suggested that using Creatine
Monohydrate may not be an effective supplement in combination with aerobic tasks.

3. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 3
Effects of Creatine Monohydrate and Exercise in Rats
For athletes and exercise enthusiasts alike, there have always been debates regarding the use of
dietary supplements in conjunction with exercise. There is a constant struggle to determine
which supplements, if any, are the best to take to enhance strength and general athletic ability.
One of the many hotly debated supplements is that of that of creatine monohydrate to enhance
strength, endurance, and aid in the recovery process. Since its introduction in the late 1980's and
early 1990's, creatine monohydrate has been through a series of praise and criticism. Hailed as a
safe way to build solid muscle by some through loading and maintenance, and condemned as a
dangerous chemical to the kidneys by others, creatine monohydrate has been in the spotlight for
both good and bad reasons. In an attempt to observe the effects creatine monohydrate may or
may not have on the body in conjunction with exercise, this study tests its effects, if any, in lab
rats.
With the focus being on strength, endurance, and any positive benefits of creatine
monohydrate (CrM), a study of CrM and Parkinson's disease proved very useful. The study
showed that when give CrM, patients with Parkinson's disease expressed an increase in not only
brain and muscle creatine, but an increase in and improvement in muscular performance (Hass,
Collins, and Juncos, 2007). This sort of study opens the door for the benefits of CrM
supplementation, for example, Hass, Collins, & Juncos (2007) took twenty patients with
Parkinson's disease and randomized them so one group would receive CrM supplementation and
resistance training, while the other group was given a placebo and resistance training. The study
showed that the muscular improvement for the CrM group was significantly greater than that of
the placebo group. The results of this study showed that, while resistance training did in fact
benefit both groups, the groups that underwent CrM supplementation improved at a significantly

4. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 4
greater level. This study gave the researchers some insight into the potential benefits of CrM
supplementation on the body.
In a study of the effects of CrM supplementation and push-up performance by U.S. Army
volunteers (Armentaro et al., 2007) it was found that even though participants were given CrM
supplementation, their push-up performance was not improved any more than the group absent
of CrM supplementation. In a similar study by Mirzaei, Rahmani-Nia, Salehi, and Rahimi (2012)
the researchers set out to test the negative effects of CrM supplementation on male wrestlers.
Thirty-one, college-aged males were randomly selected to be in either an experimental group,
which was given CrM supplementation or a control group, given only a placebo. Once given a
loading period of CrM or the placebo, the wrestlers would then be given exhaustive tests to see
what effects CrM produced. What they found was that the CrM supplementation group had more
negative side effects, whereas the placebo group was not negatively affected. One animal study
though, dealt with the interaction of CrM in anaerobic and aerobic exercises in rats (Boyadjiev,
Popov, & Delchev, 2007). The study showed that rats that were supplemented with CrM had
significant improvement in athletic performance, compared with those that received no CrM. Our
thinking is that when given CrM, rats can perform athletic tasks better than those not given CrM.
These studies have led to this idea that CrM supplementation may be a valid process in aiding
athletic performance. With these studies in mind, our hypothesis is that rats given CrM
supplementation will outperform rats given no CrM supplementation, such that, those given CrM
will have more recorded revolutions on a running wheel than those given no CrM. Creatine rats
will run more revolutions in a running wheel.

5. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 5
Method
Design
We conducted a 2 x 3 between subjects design experiment. The independent variable was
the group receiving Creatine and the dependent variable was the number of revolutions recorded
on a running wheel in a span of one hour, with intervals at five, 15, 30, 45, and 60 minutes. After
three days of testing, we found that there was a main effect on the experimental group, with more
revolutions recorded than the control group. However, the largest difference was on the first day
when no Creatine was given to either groups.
Participants
The subjects will be fifteen adult, female Long-Evans rats. There will be two groups: an
experimental group consisting of eight rats and a control group consisting of seven rats. Their
weight ranged from 249-354g. With the control group averaging a weight of 303.29g and the
experimental group averaging a weight of 303.75g. The experimental group will be given
creatine monohydrate and Jif® Creamy Peanut Butter and the control group will only receive
Jif® Creamy Peanut Butter. The rats were kept on an ad libitum diet.
Materials and Procedures

6. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 6
The materials included creatine monohydrate from Optimum Nutrition, Jif® Creamy
Peanut Butter, a stainless steel running wheel from Lafayette Instruments Company in Lafayette,
IN with attached cage and battery operated counter. The independent variable is the presence of
creatine monohydrate (CrM) with two levels 0 and .08g/kg in the rat's diet and the dependent
variable is athletic performance, measured by revolutions in a running wheel in one hour.
Day one rats were habituated to their environment for 15 minutes in order to become
familiar with the running wheel, measured at the sixty minute interval. On day two, a baseline of
athletic performance was established for each rat, such that, each rat was given sixty minutes of
free running in the wheel with revolutions recorded at 15, 30, 45, and 60 minutes. The number of
revolutions by the rat was recorded to establish individual baselines. The next five days, rats in
the experimental condition received loading doses of CrM. Loading doses were used to replicate
how athletes take CrM, such that, the rats were given four times the regular dose of CrM mixed
in with peanut butter each day. During these days, the experimental group was given 96mg of
CrM mixed in with approximately a half a teaspoon of Jif® Creamy Peanut Butter (JCPB).
During these days, the control group was given the same amount of JCPB (approximately one-
half teaspoon) but no CrM. Rats were also given maintenance doses on testing days, again to
mirror how athletes supplement. Maintenance doses were only 24mg of CrM mixed with 1/2
teaspoon of peanut butter one hour before testing in the running wheel. The mixing process for
the experimental group involved weighing out the CrM, once measured, it was then poured into a
plastic soap dish to be mixed with the JCPB.
In order to give the experimental group the correct amount of CrM, dosage was
determined by the average weight of the experimental rats (303.53g). Once average weight was
established, the rats were given a dose of 0.08g/kg based on two prior studies of rats and CrM,

7. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 7
one using 0.07g/kg and one using 0.09g/kg. This came out to be 24.3 mg, so it was rounded to
24mg for the maintenance dose and multiplied by four for the loading does. During these five
loading days, neither group of rats were run. Only on the following two test days, were both
groups of rats run in the running wheel.
During the first day, both groups were habituated with the running wheel for fifteen
minutes. The second day consisted of a running wheel pre-test to establish baseline athletic
performance for both groups. The first five rats were given a half teaspoon of JCPB, but no CrM.
There were only five rats running wheels available, so rats were ran in three groups of five. The
first rat is placed in the wheel closest to the back of the room, each consecutive rat is placed in
the following wheel respectively. Once the cart is in the testing room, take the rat that goes in the
running wheel out by hand and place it in the cage attached to the running wheel. Turn
revolution counter on and set to zero. Then follow the same procedure with each consecutive rat.
Once this has been done, give the JCPB to the next group of five rats. After five minutes, begin
recording revolutions, starting with wheel one. Take a moment in between wheels to mimic the
time it took to place rats in running wheels. Rerecord revolutions at intervals of 15, 30, 45, and
60 minutes. To do so, push the store button and record revolutions. After sixty minutes has
ended, remove rats from the wheels. Once removed, change chips in cage and below running
wheel, then return rats to cages in the rat storage area. After this time, start the next group of rats.
Once the pre-testing days are over and revolutions are recorded, it is time to begin the
CrM loading days. During the five CrM loading days, neither groups of rats are to be exercised,
instead the experimental group will begin to receive the 96mg of mixed CrM and JCPB, while
the control group is given the equivalent amount of JCPB. After the five days of loading have
ended, the final two days will consist of running wheel testing. During these two days, the rats

8. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 8
will be tested in the same way they were during the pre-testing. Such that, they will be split into
three groups of five and run for 60 minutes with revolutions at five, 15, 30, 45, and 60 minutes.
However, during these testing days, the experimental group will receive a maintenance dose of
CrM and half teaspoon of JCPB with 24mg of CrM, one hour prior to testing. The control group
will continue to receive one half teaspoon of JCPB and no CrM , one hour prior to their testing.
Both the control and experimental group will be tested on the running wheel, with revolutions
recorded for one hour on both test days. Two test days are used to minimalize variablitity and to
promote repeated measures.
Results
Data were analyzed with IBM SPSS Statistics 20. Running wheel revolutions recorded at
the five minute point a 2 x 3 (treatment group [control, creatine] x Day [Day 1, Day 2, Day 3])
ANOVA. This revealed a main effect of the treatment group, F(1,13) = 6.976, p = .020, np
2 =
.349, with the Creatine group having more revolutions than the control (see figure 1). This
difference was strongest (most significant) on day one, when the rats received no Creatine. There
was no significant interaction between treatment group and control group. There was also no
interaction between day and group, group and day. While the table on figure one seems
promising, it can be misleading because the main issue is that there was already a significant
difference present on the first day. While on the following two days, the CrM group's athletic
performance decreased, such that number of revolutions decreased as the control group's
increased.

9. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 9
Discussion
Influenced by the large amount of claims praising Creatine for its role in abetting athletic
performance, we hypothesized that rats given Creatine would outperform rats not given Creatine,
such that those given Creatine would have more running wheel revolutions recorded in one hour
than those not given Creatine. Our results did not support this hypothesis; rats that were given
Creatine recorded more running wheel revolutions on the first day, but there was a significant
difference evident between the two groups on the first day. While it seemed that Creatine was the
reasons for more revolutions, this was not the case upon further analyses because there was
already a significant difference in athletic ability between groups on the first day. This result was
contrary to what we expected based on previous research that showed Creatine supplementation
improved performance in sports with reported high-intensity exercise bouts (Hershel & Derave,
2007). With research like this, we thought that Creatine supplementation would provide
significant effects for our experimental group of rats. CrM had been recognized as a useful
supplement in aerobic and anaerobic exercise (Araujo et al., 2012). Giving insight to our idea
that the experimental group would complete more revolutions than the control group. However,
our hypothesis was not supported by our data. While previous research focused on the effects of
CrM and building muscle (Cooper, Naclerio, Allgrove, & Jimenez, 2012), other animal studies
focused on more cardiovascular based exercises (Gallo et al., 2006). While we found some
studies that showed the desired effects of CrM, we also found some that failed to do so. One of
the biggest differences in our study was the amount of time in which we supplemented the rats.
We assumed that we did not give them a long enough time span for CrM to take effect, but
counter research found that even when participants were given CrM over a month, the effects
were not significant (Syrotuik et al., 2001).

10. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 10
Our study differed from other research by task and length of supplementation time which
could explain the lack of a significant effect of CrM. CrM may not have an effect on running
wheel exercise, but it may on other tasks. It was a between-subjects study, intending to show the
effects of CrM on our experimental group. While our study was high in internal validity, it still
managed to differ from other studies due to the reduced time of CrM supplementation. Another
important factor that could have limited our study is how we measured their performance. Rather
than testing them in a way more similar to previous tests, we allowed them to run or not run, this
was a limitation because the task may not have been a good one to test on rats.
The best way to test the effects of CrM would be to mirror prior experiments more
closely and to replicate how humans take CrM. Therefore, it would be more beneficial to
administer a loading and maintenance dose of more than one week. Another way to improve this
experiment would be to use a different test than running wheel revolutions. However, this
experiment makes it more important to continue CrM studies to what tasks it can best affect.

11. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 11
References
Amital, D., Tali, V., Rubinow, A., & Levine, J. (2006). Observed Effects of Creatine
Monohydrate in a Patient with Depression and Fibromyalgia. American Journal of
Psychiatry, 163(10).
Araujo M, Junior R, Moura L, Junior M, Dalia R, Sponton A, Ribeiro C, Mello M. (2012
October 27). Creatine Supplementation in Exercised Rats: Effects on the Aerobic
Capacity. Retrieved from Open Journal of Animal Sciences.
Armentano, M. J., Brenner, A. K., Hedman, T. L., Solomon, Z. T., Chavez, J., Kemper, G. B.,
Salzberg, D., Battafarano, D. F., & Christie, D. S. (2007). The Effect and Safety of Short-
Term Creatine Supplementation on Performance of Push-Ups. Military Medicine, 172,
312-316.
Berti, S. L., Nasi, G. M., Garcia, C., Castro, F. L., Nunes, M. L., Rojas, D. B., Moraes, T. B.,
Filho, C. S. D., Wannmacher, C. M. D. (2011). Pyruvate and creatine prevent oxidative
stress and behavioral alterations caused by phenylalanine administration into
hippocampus of rats. Metabolic Brain Disease, 79-89. doi: 10.1007/s11011-011-9271-9
Boyadjiev N, Popov D, Delchev S. (2007 Dec 1). Exercise Performance and Muscle Contractile
Properties After Creatine Monohydrate Supplementation in Aerobic-Anaerobic Training
Rats. Retrieved from Journal of Sports Science & Medicine.
Cooper R, Naclerio F, Allgrove J, Jimenez A. (2012 July 20). Creatine Supplementation with
Specific View to Exercise/Sports Performance [an update]. Retrieved from JISSN.

12. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 12
Hass, C. J., Collins, M. A., & Juncos, J. L. (2007). Resistance Training with Creatine
Monohydrate Improves Upper-Body Strength in Patients with Parkinson Disease: A
Randomized Trial. Neurorehabil Neural Repari, 21(107), 107-115. doi:
10.1177/1545968306293449
Lee, C. L., Lin, J. C., & Cheng, C. F. (2012). Effect of creatine plus caffeine supplements on
time to exhaustion during an incremental maximum exercise. European Journal of
Sport Science, 12(4), 338-346. doi: 10.1080/17461391.2011.573578
Kolling, J. & Wyse, A. T. S. (2010). Creatine prevents the inhibition of energy metabolism and
lipid peroxidation in rats subjected to GAA administration. Metabolic Brain Disease,
331-338. doi: 10.1007/s11011-010-9215-9
Mirzaei, B., Rahmani-Nia, F., Salehi, Z., & Rahimi, R. (2012). Effects of Creatine Monohydrate
Supplementation on Oxidative DNA Damage and Lipid Peroxidation Induced by Acute
Incremental Exercise to Exhaustion in Wrestlers. Department of Exercise Physiology,
Faculty of Physical Education and Sport Sciences, University of Guilan, Rasht, Iran, 30-
38. doi: 613.29:796.015.54:796.81-055.1
Syrotuik DG, Game AB, Gillies EM, Bell GJ. (2001 Dec). Effects of Creatine Monohydrate
Supplementation During Combined Strength and High Intensity Rowing Training
Performance. Retrieved from PubMed.gov.

13. EFFECTS OF CREATINE MONOYDRATE AND EXERCISE IN RATS 13
Figure 1: Number of Running Wheel Revolutions in one hour across three
days.