The Effects of Habitual Caffeine Consumption on Stress in Lab Mice

Caffeine in the form of soda, coffee, and supplements is widely consumed by many people on a daily basis and in some cases has been linked to feelings of psychological stress. This experiment investigated the effect of habitual caffeine consumption on cortisol levels as they relate to psychological stress in lab mice. It was hypothesised that if caffeine was administered in four different levels (control, low, medium, and high) and tested for its effect on cortisol levels, then the high caffeine level group would have the highest cortisol levels and thus the highest stress levels. Groups of ten mice were administered caffeine daily via a natural caffeine supplement powder (guarana) dissolved in their water daily over a period of one week. Mice were separated into four glass tanks with a constant supply of food, spring water, a dark-light cycle, and ample shelter. Faecal samples were collected, and faecal assay testing was performed to determine cortisol concentrations. A single anova with Tukey\’s multiple comparisons test was performed and averages were calculated for each caffeine consumption group. It was found that there were no statistically significant differences in cortisol levels between each of the caffeine level groups.
In modern society, caffeine consumption is normalised and considered a part of everyday life. For decades, people have habitually consumed caffeine in both large and small amounts, from a cup of coffee to start their morning to multiple highly caffeinated energy drinks daily.[1] Studies have shown that caffeine is a prominent central nervous system (CNS) stimulant, and has been found to increase blood pressure as well as typical endocrine responses to stress such as glucocorticoid and catecholamine.[2] Previous studies have also linked increased caffeine consumption with heightened feelings of anxiety and depression in adults.[3] Furthermore, increased habitual caffeine intake has been proven to disrupt sleep cycles in adolescents and cause harmful side effects such as increased stress levels.[4]
Paullinia cupana (guarana) is a Brazilian plant with an extremely high natural caffeine content of 6%. The seeds of the guarana plant can be powdered and dissolved in water to be used as a natural caffeine supplement. When powdered, guarana has a caffeine content of 220 mg of caffeine/ 1 g of powder.[5]
This study asked the question: What is the effect of habitual caffeine consumption on stress in lab mice? This question was tested by examining cortisol, a stress hormone that is secreted by the hypothalamus-pituitary gland adrenal axis (HPPA), as a biomarker of psychological stress,[6] through faecal assay testing. By separating lab mice into four equal groups of ten, with each group receiving a different amount of caffeine in their water supply for one week via guarana powder, this study was able to obtain a good understanding of the effect of caffeine consumption on stress levels in mice.
By using mice, who share many common genetic features to humans, in this study, future research could be done to demonstrate the effects of caffeine consumption on anxiety in adults when put under similar circumstances. Furthermore, mice were chosen as the test subjects in this study in an attempt to control any outside stressors that could influence the subject’s overall cortisol concentrations. A controlled environment for the mice was created; what was consumed and any outside stimuli to which they were exposed were controlled. Whereas in a study using human participants, outside stressors such as school and work, and caffeine consumption outside of the study cannot be controlled.[7] Without being able to control these outside stressors, whether or not caffeine consumption was solely responsible for increased cortisol levels could not be determined. These factors served to answer the question: what is the effect of habitual caffeine consumption on stress in lab mice?
Materials and Methods
In this study, 40 BALB/c female lab mice aged 10 weeks at the beginning of the study were used as subjects. The mice were randomly separated into four groups of ten and assigned a caffeine consumption level. The mice were kept in four separate locked tanks with proportional amounts of aspen bedding, food pellets, and a constant supply of spring water via multiple glass water bottles. Ample space for play and exercise as well as an in-tank shelter were provided in each tank. The environment in each tank was kept the same to the greatest possible extent and followed the guidelines established in the Guide for the Use and Care of Laboratory Animals Eighth Edition.[8] The mice were weighed by a digital scale once at the beginning of the study and once again at the conclusion, and visually observed daily for any visible changes in the weight.
Ten mice received regular, unaltered spring water as a control, ten mice were assigned to the “low” group, receiving 0.5 mg of caffeine/kg of body weight, ten mice were assigned to the “medium” group, receiving 1.0 mg of caffeine/kg of body weight, and ten mice were assigned to the “high” group, receiving 1.5 mg of caffeine/kg of body weight.[9] The mice were administered these levels of caffeine via guarana powder (220 mg caffeine/1 g), a natural caffeine supplement, in their water daily over a period of one week.[9] The “low” group was given 2.5 mg of guarana powder daily, the “medium” group was given 5.0 mg of guarana powder daily, and the “high” group was given 7.5 mg of caffeine powder daily. Figure 1 shows how much caffeine each group received.
Caffeine Administered Daily Via Guarana Powder in Spring Water

Caffeine per Day (mg/kg of body weight) Control
(0 mg/kg)
(0.5 mg/kg)
(1.0 mg/kg)
(1.5 mg/kg)
Guarana Powder in Water Daily (mg) 0 mg 2.5 mg 5.0 mg 7.0 mg

[1](Figure 1) Created by Aubrey Chrisenbery
At the end of the one-week testing period, faecal assay testing was performed. Cortisol samples were collected in the form of faecal matter instead of blood or saliva to further eliminate any outside stressors that would have been involved in collecting these types of samples. In blood or saliva assay testing, the mice would have been transported to a local veterinarians office and had their blood/saliva extracted. This would have potentially caused immense stress for the mice as they were separated and transported. Choosing faecal assay testing was the superior method in this experiment because it allowed the mice to remain in a controlled environment while samples were collected.
The samples were collected in a biosafety level 1 lab, dehydrated and powdered, and then moved to a biosafety level 2 lab for cortisol testing. The 0.2 g dried sample of faecal matter was mixed with 2 mL of ACS grade ethanol. The samples were then mixed using a plate shaker and put through a centrifuge at 5,000 rpm for 15 minutes at 4°C. One mL of the supernatant in each sample was then extracted and put into a clean microcentrifuge tube and evaporated using a SpeedVac for ~2.5 hours. The remaining contents of the samples were then dissolved with ethanol and an assay buffer, and 50 µL (microlitres) of each sample was pipetted into the antigen-coated wells of the cortisol assay kit.
Standard dilutions were performed by adding 450 µL of the assay buffer and 50 µL of the cortisol stock into a test tube, creating a concentration of 3,200 pg/mL (picograms per millilitre). Six more dilutions were then created with concentrations of 1,600 pg/mL, 800 pg/mL, 400 pg/mL, 200 pg/mL, 100 pg/mL, and 50 pg/mL respectively. These dilutions were pipetted into the wells of the assay kit in duplicates. 50 µL of the assay buffer, 25 µL of the cortisol conjugate, and 25 µL of the cortisol antibody were then respectively added to each of the wells. The plate was then covered with a plate sealer and placed on a plate shaker for one hour. Each well in the plate was then washed four times with 300 µL of the wash buffer followed by 100 µL of the Tetramethylbenzidine (TMB) substrate. The plate was then sealed again and allowed to incubate at room temperature for 30 minutes. After the period of incubation, 50 µL of the stop solution was added to the wells. The resulting coloured wells were then put through a colorimetric microplate reader to measure the optical density and cortisol concentrations, and the standard curve was calculated.
Maximum cortisol concentration, as determined by the standard curve, was about 3,200 pg/mL. As seen in Figure 2, the average cortisol concentration for the control group was 1977.83 pg/mL, and the standard deviation was 485.49 pg/mL. The average cortisol concentration for the low caffeine group was 2103.15 pg/mL, and the standard deviation was 457.33 pg/mL. The average cortisol concentration for the medium group was 1896.19 pg/mL, and the standard deviation was 855.58 pg/mL. Finally, the average cortisol concentration for the high group was 1868.45 pg/mL, and the standard deviation was 501.10 pg/mL. A one-way ANOVA with Tukey’s multiple comparisons test (Figure 3) was performed to confirm the accuracy of the results, and it was found that there was no statistically significant difference between the groups.
([2]Figure 2) Created by Aubrey Chrisenbery
The mean cortisol concentration for each caffeine level group was from about 1750 pg/mL to 2250 pg/mL, with the medium caffeine level group having the highest average cortisol concentration, and the high caffeine level group having the lowest average cortisol concentration.
One-Way ANOVA With Tukey’s Multiple Comparisons
Cortisol Consumption Levels (mg/kg of body weight)
(Figure 3) Created by Dr Christopher Lupfer
It was observed that the cortisol levels for most of the mice fell between 1000 pg/mL and 2500 pg/mL. One mouse in the medium caffeine level group had an unusually high cortisol level when compared to the other mice in its group. This may have been a result of outside exposures to stress or the temperament of the individual mouse.
There are several implications that can be made by the lack of statistical significance in the results of this study. For example, in other studies performed under similar conditions it was found that caffeine intake greatly increased the stress levels of mice when hormone levels were measured via blood drawn from the mice. It would be interesting to research if, in mice, cortisol does not metabolise as strongly into faecal matter as compared to blood or urine samples. Furthermore, the results should not be taken to mean that caffeine does not affect psychological feelings of anxiety, but it does mean that perhaps habitual caffeine consumption does not cause cortisol levels to become elevated in lab mice.
There were several limitations throughout this experiment. Weeks prior to caffeine administration and sample collection, one mouse in the “high” tank was attacked by the other mice in the tank. As a result, this mouse lost half of its tail and was not included in the study. A veterinarian was consulted and the mouse was moved to an isolated tank for the remainder of the experiment. Thus one group only had nine subjects instead of ten. Mice from the “high” tank were then mixed into other tanks to prevent another incident like this from occurring. It is important to note that these mice had not yet received any caffeine. In the first trial, another limitation occurred due to a pipetting error. One row of the assay plate did not receive any of one chemical and another row received double of one chemical. There was no way to know which chemical was pipetted into two rows and this error skewed the results of the assay plate, and the experiment was conducted a second time. No results from the first trial were included in the results or statistical analysis.
In the second trial, after twelve hours of faecal collecting, not enough of each sample was able to be collected. At least half of the 0.2 g sample required for the cortisol assay kit was collected from each subject during this time, but in order to obtain the remaining amount of faecal matter needed 22 of the subjects were left isolated overnight in individual plastic boxes. They were left in large, individual locking tanks and sorted by caffeine level groups for about 36 hours due to unforeseen winter weather conditions that prohibited me from removing them from isolation sooner. Holes were drilled into the boxes to ensure sufficient airflow, and food, spring water, and bedding were left in each of the boxes. After this period of isolation, all remaining faecal samples needed were collected. However, over the course of the time, they were left isolated, the subjects chewed holes in the lids of the plastic boxes and escaped into the interior of the locked tank. As a result, some of the subjects were found in different boxes than they were placed. Faecal samples were still collected from each of the assigned boxes rather than the interior of the large tank to make the samples as accurate as possible. However, there is no sure way to know if all of the faecal matter in each box was from the mouse originally assigned to that box. However, the mice that were intermingled were all a part of the same caffeine level group. It is also possible that as a result of being left isolated and in a different environment for over 24 hours that subjects were more stressed out than normal, and as a result their cortisol levels were elevated, causing the results to look as though all of the mice were equally as stressed.
After calculating cortisol concentration levels for the second trial of this experiment, one mouse in the medium caffeine level group was found to have extremely elevated cortisol levels when compared to the other mice in its group and across the entire experiment. This may have been a result of the aforementioned stress of being left in a different environment overnight, a pipetting error when conducting the assay kit, or the temperament and baseline anxiety levels of that individual mouse. While the anomaly was noted, this outlier was included in the final analysis and average cortisol concentrations of the caffeine level groups.
In the ongoing research in this field, the results of this experiment are important to include. This experiment experienced numerous obstacles and difficulties that exposed the mice to unanticipated stressors. While this may have caused the average cortisol concentrations of the caffeine level groups to be very similar, it may perhaps give future researchers a good idea of the effect of habitual caffeine consumption on adults who experience moderate to high-stress levels on a daily basis. In the future, new hypotheses could be drawn such as: what is the effect of habitual caffeine consumption on stress in adults who experience clinically diagnosed anxiety? This hypothesis could be tested in the same manner as this experiment, but by using subjects that experience anxiety on a daily basis and comparing their average cortisol concentrations before and after habitual caffeine consumption.
After conducting this experiment, there are several areas for expansion and future studies. If I were to do this experiment again, I would expand a bit and possibly try removing the caffeinated water from the mice’s tanks and replacing it with unaltered spring water for a period of 24 to 48 hours before attempting faecal collection. I would also consider expanding the sample size of each caffeine level group to reduce standard deviation, while also potentially testing over a longer or shorter period of time. Improvements such as this could help to alleviate anomalies such as the outlier in the medium caffeine level group in this experiment. The lack of caffeine after habitually consuming it for a week may actually increase stress levels more than just consuming caffeine. I would also possibly test a different stress hormone, such as corticosterone, to see if there are any differences in stress hormone levels when it comes to habitual caffeine consumption.
I would like to extend my gratitude to the Whitmire Foundation for making this project possible. I would also like to thank Taconic Biosciences for their gracious donation of 40 BALB/c mice, Arbor Assays for their donation of my second Cortisol ELISA Assay Kit, and Pittsburg State University for their donation of centrifuge vials. I would especially like to thank Dr Christopher Lupfer for allowing me to use his facilities and equipment, and by extension Missouri State University. Furthermore, I would like to extend my thanks to Karisa Boyer, who advised my project and stayed with me for hours on end to complete assay protocols. Finally, I would like to thank Laheather Fisher and Timothy Oster, who allowed me to use their equipment and assisted in numerous other ways.

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  1. Figure 1: Table created by Aubrey Chrisenbery. This table demonstrates the amount of caffeine administered per day and the corresponding amount of guarana powder needed to administer that level of caffeine.
  2. Figure 2: (graph created by Aubrey Chrisenbery): This graph demonstrates the mean cortisol concentrations between the four testing groups.
  3. Figure 3: (created by Dr Christopher Lupfer): This figure shows the concentrations of each individual faecal sample from each mouse when run through a one-way ANOVA with Tukey\’s multiple comparisons test.

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