Journal of Neurotrauma

39:1339–1348 (October 2022)

Mary Ann Liebert, Inc.
DOI: 10.1089/neu.2022.0083

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ORIGINAL ARTICLE FLUID BIOMARKERS

Effect of Player Position on Serum Biomarkers

during Participation in a Season of Collegiate Football
Linda Papa,1,2,* Alexa E. Walter,3 James R. Wilkes,4 Hunter S. Clonts,1 Brian Johnson,5 and Semyon M. Slobounov4

Abstract
This prospective cohort study examined the relationship between a panel of four serum proteomic bio-
markers (glial fibrillary acidic protein [GFAP], ubiquitin C-terminal hydrolase-L1 [UCH-L1], total Tau, and neu-
rofilament light chain polypeptide [NF-L]) in 52 players from two different cohorts of male collegiate
student football athletes from two different competitive seasons of Division I National Collegiate Athletic
Association Football Bowl Subdivision. This study evaluated changes in biomarker concentrations (as indi-
cators of brain injury) over the course of the playing season (pre- and post-season) and also assessed bio-
marker concentrations by player position using two different published classification systems. Player
positions were divided into: 1) speed (quarterbacks, running backs, halfbacks, fullbacks, wide receivers,
tight ends, defensive backs, safety, and linebackers) versus non-speed (offensive and defensive linemen),
and 2) ‘‘Profile 1’’ (low frequency/high strain magnitudes positions including quarterbacks, wide receivers,
and defensive backs), ‘‘Profile 2’’ (mid-range impact frequency and strain positions including linebackers,
running backs, and tight ends), and ‘‘Profile 3’’ (high frequency/low strains positions including defensive
and offensive linemen). There were significant increases in GFAP 39.3 to 45.6 pg/mL and NF-L 3.5 to
5.4 pg/mL over the course of the season ( p < 0.001) despite only five players being diagnosed with concus-
sion. UCH-L1 decreased significantly, and Tau was not significantly different. In both the pre- and post-
season blood samples Tau and NF-L concentrations were significantly higher in speed versus non-speed
positions. Concentrations of GFAP, Tau, and NF-L increased incrementally from ‘‘Profile 3,’’ to ‘‘Profile 2’’
to ‘‘Profile 1’’ in the post-season. UCH-L1 did not. GFAP increased (by Profiles 3, 2, 1) from 42.4 to 49.6 to
78.2, respectively ( p = 0.051). Tau increased from 0.37 to 0.61 to 0.67, respectively ( p = 0.024). NF-L increased
from 3.5 to 4.9 to 8.2, respectively ( p < 0.001). Although GFAP and Tau showed similar patterns of elevations
by profile in the pre-season samples they were not statistically significant. Only NF-L showed significant dif-
ferences between profiles 2.7 to 3.1 to 4.2 in the pre-season ( p = 0.042). GFAP, Tau, and NF-L concentrations
were significantly associated with different playing positions with the highest concentrations in speed and
‘‘Profile 1’’ positions and the lowest concentrations were in non-speed and ‘‘Profile 3’’ positions. Blood-based

1
Department of Emergency Medicine, Orlando Regional Medical Center, Orlando, Florida, USA.
2
Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada.
3
Department of Neurology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
4
Department of Kinesiology, Pennsylvania State University, University Park, Pennsylvania, USA.
5
Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

*Address correspondence to: Linda Papa, MDCM, MSc, Department of Emergency Medicine, Orlando Regional Medical Center, 86 West Underwood (S-200), Orlando, FL 32806,
USA E-mail: lpstat@aol.com

ª Linda Papa et al., 2022; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (CC-BY) (http://
creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.

Correction added on February 16, 2023 after first online publication of September 29, 2022: The article reflects Open Access, with copyright transferring to the author(s),
and a Creative Commons License (CC-BY) added (http://creativecommons.org/licenses/by/4.0/).

1339
1340
biomarkers (GFAP, Tau, NF-L) provide an additional layer of injury quantification that could contribute to a
better understanding of the risks of playing different positions.
Keywords: athletes; biomarkers; concussion; football; GFAP; mild traumatic brain injury; neurofilament;
proteomics; sports; subconcussion; Tau; UCH-L1
Introduction
Nearly 8 million students who currently participate in
high school athletics and the more than 480,000 who
compete as National Collegiate Athletic Association
(NCAA) athletes1 in the United States are at risk for con-
cussive and subconcussive injuries.2 Numerous studies
have documented that both clinically diagnosed concus-
sive and repetitive subconcussive injuries induce similar
changes in brain structure and functions.3,4 The additive
effect of subconcussive impacts has the potential for
long-term deleterious effects on brain function and neu-
rodegeneration in select individuals.5-7 These concerns
are leading researchers to assess concussion risk and to
estimate contact exposure.
It appears that the location, frequency, and magnitude
of head impacts in football differ by position, with spe-
cific player positions at higher risk of concussion and
long-term sequelae.7–11 In a study of the effects of play-
ing position in former college and professional athletes,
white matter integrity, functional neural recruitment,
and concussion history depended on career duration and
playing position.9 Recent studies have stratified players
into speed and non-speed positions, with speed players
building up momentum prior to tackling or being tackled,
and non-speed players engaging other players before sig-
nificant momentum can be generated. Therefore, speed
players encompass quarterback, running back, halfback,
fullback, wide receiver, tight end, defensive back, safety,
and linebacker positions and non-speed players include
all defensive and offensive linemen.11
More recently, Karton and colleagues employed a
novel method for quantifying repetitive head impacts
using a biomechanical measurement method that incor-
porated frequency of impact, tissue strain magnitude
and time interval between impacts to measure exposures
specific to professional football player field positions.10
They evaluated 3439 head impacts for eight player
positions during 32 regular seasons and divided player
positions into three profiles. ‘‘Profile 1’’ positions were
exposed to low impact frequency of proportionately
higher strain magnitudes within 25- to 30-min time inter-
vals and included quarterbacks, wide receivers, and
defensive backs. ‘‘Profile 2’’ consisted of all levels of
magnitude of mid-range impact frequency and intervals
(approximately 13 min) and included linebackers, run-
ning backs and tight ends. ‘‘Profile 3’’ positions experi-
enced the highest frequency of head impacts within short
time intervals of (approximately 6 to 7 min) of predomi-
PAPA ET AL.
nately low strain magnitude and included offensive and
defensive lines.10 Tight ends had some overlap between
Profiles 2 and 3. Overall, there is growing evidence sug-
gesting differential vulnerability of football players to
head acceleration events a function of player position,
but neural underpinning of this effect is poorly understood.
Like other blood tests used in medicine, a blood test
for traumatic brain injury (TBI) has the potential to pro-
vide invaluable information about the severity or magni-
tude of injury to the brain. Glial fibrillary acidic protein
(GFAP) is a protein found in astroglial skeleton of both
white and gray brain matter and has been used as a his-
tological marker for glial cells. Ubiquitin C-terminal
hydrolase-L1 (UCH-L1) is protein in neurons that is
involved in the addition and removal of ubiquitin from
proteins that are destined for metabolism and has been
used as a histological marker for neurons.12 Tau is an in-
tracellular, microtubule-associated protein that is enriched
in axons and is involved with axonal microtubule assem-
bly and axoplasmic transport.13 Neurofilament light chain
polypeptide (NF-L) is principally found in axons and is
part of the neuron cytoskeleton that functions to pro-
vide structural support.14 Previous studies have shown
that these blood-based biomarkers of TBI are associated
with sports-related concussion, subconcussive injuries,
and neurocognitive functioning.15–20
This prospective cohort study examined the relation-
ship between a panel of four serum proteomic biomarkers
(GFAP, UCH-L1, total Tau, and NF-L) in two different
cohorts of male collegiate student football athletes from
two competitive seasons in a Division I NCAA Football
Bowl Subdivision. This study evaluated changes in bio-
marker concentrations (as indicators of brain injury) over
the course of the playing season (pre- and post-season)
and also assessed biomarker concentrations by player posi-
tion using two different stratification methods previously
published by Karton and colleagues and by Lehman.10,11
Methods
Study population
This prospective cohort study enrolled two different
cohorts of male collegiate student football athletes from
the Pennsylvania State University from two distinct com-
petitive seasons in a Division I NCAA Football Bowl
Subdivision. This study was approved by the Pennsyl-
vania State University Institutional Review Board and
informed consent was obtained from all participants
prior to enrollment.
EFFECT PLAYER POSITION ON SERUM BIOMARKERS
Study procedures
All participants completed a comprehensive pre-season
interview which included demographic information,
medical and concussion history (self-report), and his-
tory of learning disabilities. Pre-season blood draws
were completed within 1 week before the athletic season
began (prior to any pre-season contact practices or com-
petitions) and the postseason blood draw within 1 week
after the last game of the regular season. None of the
athletes were recovering from or were diagnosed with a
concussion in the 9 months prior to the pre-season eval-
uation. A blood sample of 5 mL was placed in a serum
separator tube and allowed to clot at room temperature
before being centrifuged. The serum was placed in bar-
coded aliquot containers and stored at -70C until
transport to a central laboratory where samples were
analyzed in batches. Lab personnel conducting the bio-
marker analysis were blinded to the clinical data. All
athletic trainers, physicians, and research personnel were
blinded to the serum biomarker results.
Outcome measures
The primary outcome measure was the change in bio-
marker concentrations over the course of a season. The
secondary outcome measure was the association between
protein biomarkers and playing positions using two dif-
ferent published classification systems. Per Lehman and
colleagues, player positions were divided into either
speed versus non-speed positions or three profiles.
Speed players included quarterbacks, running backs, half-
backs, fullbacks, wide receivers, tight ends, defensive
backs, safety, and linebackers. Non-speed players inclu-
ded all defensive and offensive linemen.11
Additionally, player positions were divided into three
profiles recently developed by Karton and colleagues in
a study that used a biomechanical measurement method
that incorporated frequency of impact, tissue strain magni-
tude and time interval between impacts to measure expo-
sures.10 In this study, ‘‘Profile 1’’ positions were exposed
to low impact frequency of proportionately higher strain
magnitudes (quarterbacks, wide receivers, and defensive
backs). ‘‘Profile 2’’ consisted of all levels of magnitude
of mid-range impact frequency and intervals (linebackers,
running backs and tight ends). ‘‘Profile 3’’ positions ex-
perienced the highest frequency of head impacts within
short time intervals (offensive and defensive lines).10
Biomarker analysis
Serum samples were analyzed using the Simoa (single
molecule array) Neurology 4-plex assay kit (Quanterix,
Lexington, MA) for simultaneous measurement of
GFAP, UCH-L1, total Tau, and NF-L on the HDX Ana-
lyzer. Assays were batched to minimize variability, with
each batch run with appropriate standards and controls
to ensure reliability. The laboratory was blinded to the
1341
clinical data. All samples were analyzed in duplicate.
None of the intra-assay coefficients of variance exceeded
20%. Samples with concentrations below the level of
detection were excluded from analysis, this included one
pre-season UCH-L1 value and three post-season UCH-
L1 values. The average coefficient of variation of measure-
ment of GFAP, UCH-L1, Tau and NF-L were 7.7%,
10.2%, 6.3%, and 5.8%, respectively. Limit of detection
of GFAP was 0.211 pg/mL, UCH-L1 was 1.05 pg/mL,
Tau was 0.0146 pg/mL, and NF-L was 0.038 pg/mL.
Statistical analysis
Descriptive statistics with medians, proportions and inter-
quartile ranges were used to describe the data. For statis-
tical analysis, biomarker levels were treated as continuous
data and measured in pg/mL. Data were assessed for
equality of variance and distribution using the Shapiro-
Wilk test. Logarithmic transformations were conducted
on non-normally distributed data. Concentrations of all
four biomarkers had to be transformed. Group compari-
sons were performed using the Fisher’s exact test, analy-
sis of variance, and independent sample t-tests with
variance consideration. Correlational analysis used Pear-
son’s correlation and Spearman’s rank correlation. All
analyses were performed using the statistical software
package SPSS 27.0 (IBM Corporation).
Results
There were 52 male collegiate student football athletes
included in the study. Mean age of the enrolled athletes
was 21 years (with a range from 19 to 24) with a mean
height and weight of 75 inches and 265 pounds, respec-
tively (Table 1). The average number of years of foot-
ball experience was 11 years, and 40% had previously
experienced a concussion. There were 32 athletes in
non-speed positions and 20 athletes in speed positions.
There were no significant differences in baseline charac-
teristics between the two groups except in weight. Players
in the non-speed positions were significantly heavier
than players in the speed positions (291 vs. 224 lbs;
p < 0.001; Table 1). Five total players were diagnosed
with a concussion (9.4%), two players from the first
cohort and three players in the second cohort (Table 1).
There were no significant differences in concussion inci-
dence in the speed (n = 2; 9.5%) and non-speed (n = 3;
9.4%) players ( p = 0.99) and no significant difference
in between ‘‘Profile 1’’ (n = 1; 17%), ‘‘Profile 2’’ (n = 1;
7%) and ‘‘Profile 3’’ (n = 3; 9%; p = 0.78).
Collectively, there were significant increases in GFAP
and NF-L serum concentrations over the course of the
season for all players under study (Table 2). GFAP
increased from a median of 39.3 (interquartile range
[IQR] 31.7-56.3) to 45.6 (IQR 35.2-63.3; p < 0.001).
Similarly, NF-L increased from 3.5 (IQR 3.0-4.6) to
5.4 (IQR 3.7-7.0; p < 0.001). UCH-L1 levels
1342 PAPA ET AL.

Table 1. Characteristics of Athletes Included in the Study in Speed and Non-Speed Positions

Non-speed positions Speed positions Total

n = 20 n = 32 N = 52
[95% CI] [95% CI] [95% CI] p Value

Age 21 [20-21] 21 [20-21] 21 [20-21] 0.570

Range 19-24 18-24 18-24
Height (inches) 76 [74-77] 74 [72-76] 75 [74-76] 0.099
Weight (lbs) 291 [282-300] 224 [213-235] 265 [254-278] <0.001
Years playing football 11 [10-13] 12 [10-13] 11 [10-12] 0.759
Previous concussions 13 (41%) 8 (40%) 21 (40%) 0.598
History of ADHD 8 (25%) 2 (10%) 10 (19%) 0.283
Player positions 14 (44%) 11 (55%) 25 (48%) 0.570
Offense 18 (56%) 9 (45%) 27 (52%)
Defense
Concussion during season 3 (9.4%) 2 (9.5%) 5 (9.4%) 0.999

ADHD, attention-deficit hyperactivity disorder.

decreased significantly ( p = 0.047), and Tau levels were
not significantly different (Table 2). There were no signifi-
cant differences in post-season biomarker concentrations
between players who were and were not diagnosed with
concussion during the season (Table 3). Similarly, concus-
sions previously incurred before the current season did not
correlate with concentrations of pre-season biomarkers.
When biomarker concentrations were compared in
speed and non-speed position players,11 there were sig-
nificant differences in median levels of Tau and NF-L
between the two groups (Fig. 1). The differences were
evident in both pre- and post-season serum samples.
Median pre-season Tau concentrations were 0.63 (0.50-
0.94) in non-speed players, compared with 0.91 (0.67-
1.02) in speed players ( p = 0.032). Median post-season
Tau concentrations were 0.53 (0.37-0.87) in non-speed
players and 0.87 (0.62-1.07) in speed players ( p = 0.011).
Similarly, median pre-season NF-L concentrations were
3.4 (2.7-4.1) in non-speed players compared with 4.3
(3.3-5.1) in speed players (p = 0.033). Even more pronoun-
ced differences were seen in post-season NF-L concen-
trations with speed players having significantly higher
concentrations than non-speed players 6.7 (5.3-8.7) versus
4.7 (3.5-5.7), respectively ( p = 0.004). GFAP concentra-
tions were also higher in speed versus non-speed position
players, but the differences were not statistically signifi-
cant. Differences were not evident for UCH-L1.
Using the profiles created by Karton and colleagues,
players were divided into three profiles based on their
Table 2. Comparison of Biomarker Concentrations
Pre- and Post-Season
positions: n = 5 in ‘‘Profile 1,’’ n = 15 in ‘‘Profile 2,’’
and n = 32 in ‘‘Profile 3.’’10 In the post-season, concentra-
tions of GFAP, Tau, and NF-L increased incrementally
from ‘‘Profile 3’’ to ‘‘Profile 2’’ to ‘‘Profile 1’’ (Fig. 2).
GFAP increased from 42.4 to 49.6 to 78.2 pg/mL, respec-
tively ( p = 0.051). Tau increased from 0.37 to 0.61
to 0.67 pg/mL, respectively (p = 0.024). NF-L increased
from 3.5 to 4.9 to 8.2 pg/mL, respectively ( p < 0.001).
Although GFAP and Tau showed similar patterns of
elevations by profile in the pre-season, they were not
statistically significant. In the pre-season, GFAP con-
centrations changed from 37.7 to 37.2 to 61.4 pg/mL,
respectively ( p = 0.063) and Tau concentrations increa-
sed from 0.50 to 0.63 to 0.73 pg/mL, respectively
( p = 0.093). Only NF-L serum concentrations showed
statistically significant differences by profile, 2.7 to 3.1
to 4.2 pg/mL, respectively, in the pre-season ( p = 0.042).
Differences were not evident for UCH-L1.
Changes in biomarker concentrations from pre- to
post-season in speed versus non-speed players as well
as among the three different profiles are presented in
Table 4 and in Supplementary Figure S1 and Supplemen-
tary Figure S2. GFAP and NF-L demonstrated large
increases in speed players Compared with non-speed
players over the season. GFAP increased 31% (speed)
Table 3. Comparison of Post-Season Biomarker
Concentrations between Players Who Were and Were Not
Diagnosed with Concussion during the Season
No concussion Concussion
during season during season
n = 47 n=5
median (IQR) median (IQR) p Value

Pre-season Post-season Post-season 43.8 (35.0-63.4) 47.4 (37.3-76.4) 0.687

n = 52 n = 52 GFAP (pg/mL)
median (IQR) median (IQR) p Value Post-season 4.8 (3.0-10.6) 4.7 (2.2-5.7) 0.417
UCH-L1 (pg/mL)
GFAP (pg/mL) 39.3 (31.7-56.3) 45.6 (35.2-63.3) < 0.001 Post-season 0.66 (0.47-0.97) 0.67 (0.54-1.29) 0.310
UCH-L1 (pg/mL) 6.9 (3.1-12.7) 5.0 (3.0-9.8) 0.047 Total Tau (pg/mL)
Total Tau (pg/mL) 0.82 (0.57-0.98) 0.67 (0.49-0.97) 0.105 Post-season 5.3 (3.6-6.9) 5.9 (5.6-9.0) 0.197
NF-L (pg/mL) 3.5 (3.0-4.6) 5.4 (3.7-7.0) < 0.001 NF-L (pg/mL)

GFAP, glial fibrillary acidic protein; UCH-L1, ubiquitin C-terminal GFAP, glial fibrillary acidic protein; UCH-L1, ubiquitin C-terminal
hydrolase-L1; NF-L, neurofilament light chain polypeptide. hydrolase-L1; NF-L, neurofilament light chain polypeptide.
EFFECT PLAYER POSITION ON SERUM BIOMARKERS 1343

versus 12% (non-speed) and NF-L increased 87% (speed)
versus 45% (non-speed). A similar pattern emerged for
the three profiles. GFAP increased 44% in ‘‘Profile 1’’
players, 27% in ‘‘Profile 2’’ players, and 12% in ‘‘Profile
3’’ players. The largest increases were seen in NF-L with
rises of 165%, 62%, and 45% in Profiles 1 through 3,
respectively. UCH-L1 and Tau did not follow these pat-
terns. These changes were not statistically significant.
Discussion
This is among the first studies, to our knowledge, to
assess the association between player position in foot-
ball and proteomic biomarker concentrations in the pre-
and post-season. This study evaluated a panel of four
proteomic biomarkers of brain injury (GFAP, UCH-L1,
Tau, and NF-L) in two distinct cohorts of players in
two competitive seasons of Division I NCAA Football.
Compared with pre-season, post-season levels of GFAP
and NF-L1 increased significantly. Only five players
were diagnosed with concussions during the two foot-
ball seasons and there were no significant differences
in post-season biomarker concentrations between play-
ers who were and were not diagnosed with concus-
sions. Therefore, head impact exposures that did not
result in concussion were significant enough to raise
biomarker levels, suggesting cellular disruption from non-
concussive impacts.
In samples collected in the pre-season (prior to any
contact practices), all four biomarkers were above known
baseline levels of uninjured control patients that have
been analyzed on the same assay platform.21 Pre-season
elevations in biomarkers have been observed in other
studies in collegiate football players.15,22 Considering
the players in our cohort had an average of 11 years play-
ing experience and 41% had previously reported con-
cussions, pre-season biomarker elevations could suggest
residual circulating biomarkers from prior concussive
and subconcussive impacts over their playing career.
Subacute (weeks) and chronic (months to years) eleva-
tions in GFAP and NF-L concentrations have been
found in patients following mild to moderate TBI com-
pared with uninjured control patients with more variabil-
ity in the pattern of elevations with Tau and UCH-L1.23,24
Chronic elevations of Tau tend to occur in severe TBI
patients and UCH-L1 does not have a consistent pattern
of elevation either by severity of injury or time.23
A novel aspect of this study was the evaluation of
the effects of player position on TBI biomarkers. We
observed significant higher levels of Tau and NF-L in
speed positions such as quarterbacks, running backs,
halfbacks, fullbacks, wide receivers, tight ends, defen-
sive backs, safeties, and linebackers, compared with non-
speed players like defensive and offensive linemen.
Speed players build considerable momentum prior to
tackling or being tackled. In contrast non-speed players
usually engage other players soon after the football is
snapped, reducing momentum prior to contact. NF-L
and Tau as measures of axonal injury are much higher
in those speed layers, perhaps indicating higher ‘‘shear’’
forces to axons. Axons, however, are not injured in isola-
tion and injuries to the astroglia (GFAP) and neuronal
bodies (UCH-L1) also occur.
This is consistent with the three-tier ‘‘brain strain
exposure’’ profiles in which post-season GFAP, Tau,
and NF-L concentrations were highest in ‘‘Profile 1’’
players (quarterbacks, wide receivers, and defensive
backs) who had higher strain magnitudes impacts. Even
though they occurred less frequently, the ‘‘higher strain
magnitude’’ impact players had the highest levels of
GFAP, Tau and NF-L. Impact strain had more of an effect
on the biomarkers than the frequency of impacts and
intervals between impacts.
These incremental changes in biomarkers from highest
to lowest strain magnitude occurred not only after the
season but were evident in player samples even before
the season began. It was unexpected considering there
had been no practices or football hitting activity. Pre-
season GFAP, Tau, and NF-L demonstrated these simi-
lar changes by player position profile but only NF-L
was statistically significant. This could be a function
of the modest sample size or a function of the different
temporal profiles of each biomarker.23,25,26 For instance,
serum NF-L can remain elevated in mild TBI for up
to 5 years after injury compared with controls.23 This
supports our hypothesis that elevations in circulating
biomarker concentrations prior to season onset could

‰
FIG. 1. Boxplot comparing of median serum levels of four protein biomarkers [GFAP], ubiquitin C-terminal
hydrolase-L1 [UCH-L1], total Tau, and neurofilament light chain polypeptide [NF-L]) by player speed position.
Boxplots represent medians with interquartile ranges. (A) Pre-season serum GFAP concentrations in non-speed
vs speed positions; (B) Post-season serum GFAP concentrations in non-speed vs speed positions; (C) Pre-
season serum UCH-L1 concentrations in non-speed vs speed positions; (D) Post-season serum UCH-L1
concentrations in non-speed vs speed positions; (E) Pre-season serum Tau concentrations in non-speed vs
speed positions; (F) Post-season serum Tau concentrations in non-speed vs speed positions; (G) Pre-season
serum NF-L concentrations in non-speed vs speed positions; (H) Post-season serum NF-L concentrations in
non-speed vs speed positions.
1344
EFFECT PLAYER POSITION ON SERUM BIOMARKERS 1345

reflect prior cumulative head impact exposures includ-
ing players without prior history of concussions. Visible
signs or symptoms of neurological dysfunction may not
develop despite those impacts having the potential for
neurological injury.3,6,27 This includes studies in children
aged 8 to 13 years who play football28 as well as those
who play high school football.29
Another important consideration is that there may be
differences in the mechanism of injury between player
positions, including player’s helmet impact location.30
For instance, offensive and defensive lineman may
have a greater proportion of impacts to the front of the
helmet.7 Lowest strains consistently occur in impacts to
the crown and forehead.30 Other players may have impact
locations that contribute to greater cervical strain. The
cervical spine is particularly susceptible to injury because
of axial loading forces to the head with the neck in flexion
or extension.31,32
These results support exploring these biomarkers as
surrogates of subconcussive damage as they relate to
magnitude, location, and frequency of head impacts.
Biomarkers provide an additional layer of injury quanti-
fication that could contribute to a better understanding
of the risks of playing different positions. Moreover,
this type of biomarker-positional analysis could be used
to investigate game rule changes, player safety measures,
on-field behavior, return-to-play decisions, and enhance-
ments to equipment.33
Limitations
While these data are encouraging, there are limitations to
this study. Athletes enrolled over two non-consecutive
seasons represents a small sample of athletes. The play-
ers, however, are representative of many players in
this Division of the Football Bowl Subdivision of the
NCAA. Only five players were diagnosed with concus-
sion in the study population during the season. Addition-
ally, there were only five players in ‘‘Profile 1’’ positions.
Future studies should include a larger sample of players.
Since non-speed players traditionally weigh more than
speed players, our study is limited by the lack of informa-
tion on weight’s impact on the studied baseline biomark-
ers in non-athletes. We also did not assess the impact of
exertion, sleep and supplement use on biomarker levels.
Moreover, we did not report neurocognitive data or imag-
ing data on the players and did not correlate biomarker
levels with these measures.
We used previously published categorizations of
player position and did not report actual accelerometer
data from the players. Additionally, we have no infor-
mation pertaining to helmet standardization, nor did we
factor the type of helmet the players were using during
the study. Not all collegiate programs utilize the same
brand of helmet, and it would be interesting to study the
elevation of biomarkers from sub-concussive impacts
between different helmet technologies.
Conclusion
Serum concentrations of GFAP and NF-L increased
significantly over the course of the season despite few
players being diagnosed with concussion. Moreover, post-
season GFAP, Tau, and NF-L concentrations were sig-
nificantly associated with different playing position
profiles based on magnitude, location, and frequency
of head impacts. The highest concentrations of post-
season GFAP, Tau, and NF-L were in speed and
‘‘Profile 1’’ positions (quarterbacks, wide receivers,
and defensive backs) and the lowest concentrations
of GFAP, Tau, and NF-L were in non-speed or ‘‘Pro-
file 3’’ positions (defensive and offensive linemen).
Blood-based biomarkers provide an additional layer
of injury quantification that could contribute to a
better understanding of the risks of playing different
positions.
Funding Information
This study was supported in part by Award Number
R01NS057676 (Papa, PI) from the National Institute of
Neurological Disorders and Stroke. The content is solely
the responsibility of the authors and does not necessarily
represent the official views of the National Institute of
Neurological Disorders and Stroke or the National Insti-
tutes of Health. The funder had no role in the design and
conduct of the study; collection, management, analysis,
and interpretation of the data; preparation, review, or
approval of the manuscript; and decision to submit the
manuscript for publication.

‰
FIG. 2. Boxplot comparing of median serum levels of four protein biomarkers [GFAP], ubiquitin C-terminal
hydrolase-L1 [UCH-L1], total Tau, and neurofilament light chain polypeptide [NF-L]) by player position profile.
Boxplots represent medians with interquartile ranges. (A) Pre-season serum GFAP concentrations in three distinct
player position profiles; (B) Post-season serum GFAP concentrations in three distinct player position profiles; (C)
Pre-season serum UCH-L1 concentrations in three distinct player position profiles; (D) Post-season serum UCH-L1
concentrations in three distinct player position profiles; (E) Pre-season serum Tau concentrations in three distinct
player position profiles; (F) Post-season serum Tau concentrations in three distinct player position profiles; (G) Pre-
season serum NF-L concentrations in three distinct player position profiles; (H) Post-season serum NF-L
concentrations in three distinct player position profiles.
1346
EFFECT PLAYER POSITION ON SERUM BIOMARKERS 1347

Table 4. Change in Biomarker Concentration (Mean and Percent Difference) over the Season by Player Position

Non-Speed Speed Profile 1 Profile 2 Profile 3

n = 32 n = 20 n=5 n = 15 n = 32
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI)

GFAP
% Change 12% (5-19) 31% (2-60) 44% (-77-165) 27% (0.04-54) 12% (5-19)
Mean change (pg/mL) 5.1 (2.3-7.8) 13.1 (-1.7-27.9) 29.3 (-47.2-106) 7.8 (1.0-14.6) 5.1 (2.3-7.8)
UCH-L1
% Change 58% (-42-157) -3% (-43-37) 8% (-99-115) -7% (-56-42) 57% (-42-157)
Mean change (pg/mL) -1.6 (-3.7-0.5) -1.1 (-3.6-1.5) 0.7 (-6.6-8.1) -1.8 (-4.7-1.1) -1.6 (-3.7-0.5)
Total Tau
% Change -8% (-19-3) -1 (-14-12) 4% (-23-32) -3% (-20-13) -8% (-19-3)
Mean change (pg/mL) -0.09 (-0.16- -0.02) -0.01 (-0.13- 0.12) 0.05 (-0.24-0.34) -0.02 (-0.18-0.13) -0.09 (-0.16- -0.01)
NF-L
% Change 45% (25-65) 87% (26-149) 165% (-135-464) 62% (27-97) 45% (25-65)
Mean change (pg/mL) 1.3 (0.08-1.9) 3.3 (0.9-5.8) 6.8 (-5.1-18.6) 2.2 (1.0-3.4) 1.3 (0.8-1.9)

GFAP, glial fibrillary acidic protein; UCH-L1, ubiquitin C-terminal hydrolase-L1; NF-L, neurofilament light chain polypeptide.

This study was supported in part by NCAA Grant
#106031 ‘‘Effects of cumulative head acceleration events
(HAE) over a single athletic season on brain functional
and structural integrity.’’ The content is solely the respon-
sibility of the authors and does not necessarily represent
the official views of the NCAA. The funder had no role
in the design and conduct of the study; collection, man-
agement, analysis, and interpretation of the data; prepara-
tion, review, or approval of the manuscript; and decision
to submit the manuscript for publication.
Author Disclosure Statement
No competing financial interests exist.
Supplementary Material
Supplementary Figure S1
Supplementary Figure S2
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