The Fatigue Biomarker Story – Part II

Blog written by John Kalns


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This is the second part in John Kalns’ ongoing story about the discovery of the fatigue biomarker. We ended Part I with the explanation of cell lines and the problems researchers face with working with saliva in cell line-based experiments. And now for part two…

Getting Started with the Fatigue Experiment

So now the question is, how do you do a fatigue experiment? Well, there are several ways to go about it, but we chose to focus on the one that we thought would have the highest relevance to the U.S. Army: endurance exercises.

In a collaboration with a university, we asked eight individuals to participate in high levels of physical exertion (treadmill jogging and cycling) in a laboratory for eight hours. We collected saliva samples – before, during and after the experiment – and looked for differences.

We assumed that after 8 hours of training, these subjects would be tired; our assumption was correct. Markers of fatigue include molecular entities that appear after 8 hours of exercise, but not in an initial sample from before the start of exercise.

Over the Course of the Experiment

We took various measurements over the course of this experiment. We measured blood, saliva, and exhaled breath, as well as took surveys and muscle biopsies. For the sake of simplicity, I won’t describe the technical details here, but basically we decided to pursue two paths when searching for fatigue biomarkers.


Our first step was to examine the “usual suspects” – things people have claimed indicate fatigue (for years and, in some cases, decades). Not surprisingly, many of these changed (as expected) during this very fatiguing challenge. The problem with the “usual suspects” is that they are poor indicators of fatigue. This is mainly because the extent of change observed is relatively modest compared to the seemingly random variation in levels observed.

This is a very important concept because, ideally, you want to measure something that will unequivocally signal “fatigue”. Here’s the bottom line: the “usual suspects” may tell us something about fatigue, but it’s far from clear. They just aren’t very useful in helping people understand how they can better manage their own fatigue.


Next, we examined all low-molecular weight proteins in saliva. We looked for those molecules that were significantly increased or decreased after eight hours of exercise. While this approach may be a bit more complicated than the “usual suspects” method, it has the advantage of potentially discovering something novel.

Compared to blood and urine, saliva contains many small proteins (called peptides). We examined 5,000 or so peptides and found that most were unchanged by exercise; however, a few peptides did change. We searched for proteins that both showed the most change and were reasonably abundant in saliva. What we found was truly startling.

So, What Did We Find?

We discovered that the ratio of two small peptides was decreased by about 1,000-fold in fatigue. We also found that the variation associated with this measure was relatively modest, meaning that the *all-important* uncertainty was very low. By measuring these two peptides, we were able to make a truly objective measurement of fatigue!

Continuing the Story

Using this approach, we knew the molecular weight and several important chemical properties of the molecule of interest, but we didn’t know much else. And, to succeed in the world of proteins, you need to understand the sequence of amino acids and their modifications.

There was a lot of speculation chatter, and even a few bets were made. It seemed the most promising favorites were maybe a neuropeptide from the brain or some early inflammation marker.

In order for us to identify the sequence, we had to work for another couple of months isolating the molecule from saliva. We also collaborated with another laboratory that owned a very expensive machine that could tell us the exact molecular identity.

Wrapping Up Part II

To validate our findings, we had to synthesize the molecule and compare it to what we thought we had discovered. This is how good science is done, and I am proud of our team’s work. When we finally got the results, we were very surprised…

Up Next

Stay tuned for the final part in The Fatigue Biomarker Story! Check back soon to read part three!

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