Effects of Repetitive Subconcussive Head Impacts on Ocular-motor Function and Brain-derived Blood Biomarker
Phase: NA
Conditions: Trauma, Head
Sponsor: Indiana University



 
 
 
 
 
 
 
 
Effects of soccer heading on ocular-motor 
function, brain-derived blood biomarker, and 
neuronal activity 
 
Keisuke Kawata, PhD, Assistant Professor 
Kinesiology, 
School of Public Health, Indiana University 
1025 E. 7th Street, SPH C215 
Bloomington, IN 47401 
 
 
 
 
 
Hannah Block, PhD, Assistant Professor 
Kinesiology, 
School of Public Health, Indiana University 
 
Allison Gruber, PhD, Assistant Professor 
Kinesiology 
School of Public Health, Indiana University 
 
Zhongxue Chen, PhD, Associate Professor 
Epidemiology and Biostatistics 
Indiana University 
 
Sharlene Newman, PhD, Associate Professor 
Psychological and Brain Sciences 
Indiana University 
 
 
 
 
 

Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Inclusion/Exclusion Criteria 
4.0 
Enrollment/Randomization 
5.0 
Study Procedures 
6.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
7.0 
Study Withdrawal/Discontinuation 
8.0 
Statistical Considerations 
9.0 
Privacy/Confidentiality Issues 
10.0 Follow-up and Record Retention 
 
 

1.0 
Background 
 
In the United States, every year approximately 1.7 million people sustain a traumatic 
brain injury (TBI) with the majority (80% or 1.4 millions) of these injuries falling into the 
category of concussion or mild TBI (mTBI).1 Concussion is classified as a mild form of 
traumatic brain injury, while changes in neural function following concussions are far 
from benign. Concussed athletes frequently experience lingering neurological 
symptoms, including headache, irritability, blurred vision, and confusion.2 In respect to 
these symptoms, researchers have utilized various metrics to study brain damage, 
including brain-derived blood biomarkers, sensory systems, and neuroimaging. 
However, due to exceedingly high intra-group variability in the concussed cohort, to date 
there is no gold standard metric to gauge severity of brain damage following head 
impacts.3 
 
One possible explanation of the high variability is due to subconcussive head impact, 
defined as an impact to the head that does not cause any symptoms of concussion, 
regardless of magnitude of the impact. It, however, has the potential to cause insidious 
effects in the brain if sustained repetitively over time.4 An average athlete in contact 
sports experiences nearly a thousand subconcussive hits per season.5 While the vast 
majority of research has focused on a single impact that elicits concussion (e.g., 
disorientation, headache), many athletes are frequently exposed to subconcussive head 
impacts prior to the concussive blow.6, 7 Thus, it remains completely unknown whether 
neuronal damage and sensory deficit are caused by a single concussive blow, repetitive 
subconcussive impacts before the concussive blow, or both. 
 
Ocular-motor System 
Recently, the ocular-motor system has garnered significant scientific attention because 
of its sensitivity to various severities of brain injury.8 The ocular-motor system 
orchestrates accommodation and vergence, and their concomitant adjustment enables 
individuals to visualize an object at various distance, direction, and speed.9 Nearly 50% 
of the neural connections in the human brain, including cortical, cerebellar, and 
brainstem networks are involved in vision and eye movement functions. Since most 
head impacts cause the brain to bounce linearly and rotationally within the skull, almost 
90% of concussed individuals exhibit some form of visual dysfunction.10, 11 For example, 
a notable decline in saccades –rapid changes in eye orientation– has been observed in 
concussed boxers, football, and rugby players.12, 
13 Similarly, a near point of 
convergence (NPC), which is the closest point one can visualize an object before 
diplopia occurs, was impaired by 3-fold in concussed athletes and soldiers.14, 15 
Neuroimaging confirmed that these ocular-motor impairments were in concert with 
altered neuronal activity after concussion.16  
 
Previously, for the first time we have demonstrated that repetitive subconcussive head 
impacts could significantly impair NPC in soccer17 and football players.18 More 
intriguingly, the NPC has predicted concussions before they occurred. In our football 
study, we tracked changes in NPC in 25 players, and 3 of 25 players were diagnosed 
with a concussion (Fig 1: unpublished). There was a biphasic increase in NPC: 1) acute 
increase after 1st full-gear practice (Pads-ON1) compared with pre-practice, and 2) on 
the day of concussion, 3 players showed abnormally high NPC before the practice. Due 
to absence of concussion signs and symptoms, the players participated in the practice. 
Following moderate intensity of impacts, the 3 players became unconscious for < 30 

seconds  and  diagnosed  with  a  concussion  by  team  athletic  trainers.  Despite  it  is 
preliminary,  the  ocular-motor  function  has  the  potential  for  a  paradigm  shift  in 
concussion  diagnosis.  However,  
it  
is  
imperative  
to  
first  understand  
the  acute  effects  of 
subconcussive head impacts on the ocular-motor system. 
 
 
Blood Biomarker 
Importantly, identifying  a  biomarker  that  can  detect cumulative  risk for  developing 
neurodegenerative  disease is  a  paradigm  shifting. A Particularly Interesting New 
Cysteine Histidine-rich  (PINCH) protein is  an  adaptor  protein  involved  in  cell 
maintenance and survival of neuronal cells.19 PINCH is nearly 
undetectable in a healthy brain. However, PINCH expression 
is  upregulated  as  neuronal  axons  experience  a  high  rate  of 
strain forces, leading to disruption of tau –an axon stabilizing 
protein– from  axonal  microtubules  (Fig  2).  When  tau 
disruption occurs, PINCH binds to tau and makes it difficult to 
degrade and clear from the brain.20 Consequently, tau protein 
accumulates in  the  brain (Fig  2),  as  often  observed  in 
Alzheimer’s  disease and chronic  traumatic  encephalopathy 
(CTE).
21-23 If  PINCH  
is  detectable  
in the brain or even in the 
blood, neurons are mostly likely injured. Thus, this biomarker 
is  potentially  a  groundbreaking  because  it  carries  an  early 
sign of neurodegeneration.  
 
In  
the  preliminary  work,  we  
first  examined  plasma  PINCH  
in 
epilepsy patients, given that epilepsy is considered a form of 
neurotrauma  due  to  neuronal  hyper-excitability.  We  found 
that a robust expression of PINCH was detected in the blood of epilepsy patients while 
undetectable  in  healthy  controls  (Fig  3:  in  print).
24 Similarly,  10  out  of  16  collegiate 
football  players  showed  noticeable  increases  in  plasma  PINCH  level  at  post-season 
compared with pre-season baseline (Fig 4: unpublished). Since none of 16 athletes were 
diagnosed  with  a  concussion  during  
the  season,  
the  
increased  
level  of  plasma  PINCH 
indicates the neuronal damage from cumulative subconcussive impacts. 
 
Neuronal Activity 
Peripheral diagnostic tools for brain injury have an immense clinical implication, however 
those  methods  are  insufficient  to  truly  gauge  neuronal  dysfunction  after  head  hits. 
Electroencephalography (EEG) has produced promising results in patient with  
0
2
4
6
8
10
12
14
16
18
20
22
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
baseline
PadsOFF
PadsON1
PadsON2
PadsON3
PadsON4
Near Point Convergence (cm)
Player A
Player C
Increase
Stable
Plateau
Abnormally
High
Increase
Increase
After
Concussion
Concussion 
During 
Practice
After
Concussion
Abnormally
High
Player B
Concussion 
During 
Practice
Fig 1. The near point of 
convergence (NPC) “predicted” 
concussion before it occurred. 
Three players showed near identical 
trends. Acute NPC increase was 
observed after Pads-ON1 practice 
(full-gear practice), then remained high 
without normalized to baseline level, 
indicating slow recovery nature of the 
ocular-motor system. Player B and C 
sustained concussions during Pads-
On 3 practice, and Player A sustained 
a concussion during Pads-On 4 
practice. Both time, NPC before the 
practice was abnormally high (with no 
outward symptoms), suggesting 
particular vulnerability of the players 
on that day.  
Fig 2. PINCH binds to 
disrupted tau and 
exacerbates tau aggregation. 
P, phosphate. Figure modified 
from Kawata et al.
23  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
concussion,  with  reduced  mean  frequency  of  alpha,  reduced  power  
in  the  alpha  and 
beta frequency bands, hypercoherence between frontal regions, and decreased gamma 
frequency.
25-29 In addition to EEG, transcranial magnetic stimulation (TMS) has garnered 
significant  attention  in  scientific  society  recently,  due  to  its  non-invasive  method  to 
measure  neuronal  excitability.  However,  to  date, there is virtually  no  literature 
demonstrating  the  effects  of  minor  head  impacts  on  neuronal  activity  using  EEG  nor 
TMS. 
 
Soccer Heading Modality 
Soccer  heading  is  a  common  skill  performed  by  soccer  athletes  during  practice  and 
games. An average collegiate soccer players perform about 500 headers during a single 
season and over 3000 during the course of a career 30,  31  Research has demonstrated 
the  need  
for  
increased  awareness  of  potential  brain  
injury  associated  with  heading  
the 
soccer ball with some suggesting that the cumulative effect of soccer head impacts over 
a  career  may  lead  to  outcomes  similar  to  sustaining  multiple  concussions.32 Various 
studies have been conducted to examine the effect of an acute bout of soccer heading, 
including  Dr.  Kawata’s  previous  laboratory  at  Temple  University,  with  most  authors 
reporting  no  significant  effect  of  heading  on  the  brain  function  measures  of 
neuropsychological or balance performance (Table 1). We have tested collegiate soccer 
players post heading ball speed up to 50 mph with no significant alterations in common 
Fig 3. PINCH protein was detectable in mesial temporal epilepsy patients’ blood. 
Robust plasma expression of PINCH was observed in both medically controlled (no seizure) 
and refractory (transient seizure) epilepsy patients, while no PINCH in healthy controls. 
Fig 4. Post-season plasma PINCH was notably higher than pre-season baseline.  
10 out of 16 players showed increase in plasma PINCH at post-season. * Player 5 and 
13 did not play for the season due to surgery. Control: a subject with no history of head 
trauma. LB, linebacker; OL, offense lineman; WR, wide receiver; DL, defense lineman; 
DB, defensive back; RB, running back; TE, tight end. 
A
B
41kDa
26kDa
PINCH 
(35 -37kDa)
Healthy Control (n=5)
Medically Controlled
Medically Refractory
Epilepsy Patients (n=7)
41kDa
26kDa
51kDa
PINCH 
(35 -37kDa)
B
A
41kDa
Pre-Season
Post-Season
Pre-Season
Post-Season
1
LB
2
OL
3
WR
4
OL
5
DL
6
LB
7
DL
8
DB
Control
9
OL
10
RB
11
TE
12
LB
13
DL
14
OL
15
TE
16
WR
Control
26kDa
PINCH 
(35 -37kDa)
41kDa
26kDa
PINCH 
(35 -37kDa)
9
10
11
12
1
2
3
4
*
*
B
7
DL
8
DB
Control
15
TE
16
WR
Control
9
Co n 
tro 
l
10
11
12
13
14
15
16
1
Co n 
tro 
l
2
3
4
5
6
7
8

clinical measures (e.g., balance error scoring system, signs and symptoms checklist). 
However, sensitive modalities like blood biomarker and ocular-motor testing begin to 
unravel subclinical perturbation caused by minor head hits if sustained repetitively.17, 18, 33  
 
 
2.0 
Rationale and Specific Aims 
 
Aim 1. Determine acute effects of subconcussive head impact on plasma PINCH 
and tau expression using controlled soccer heading model. 
Hypothesis: Repetitive subconcussive head impacts will significantly increase plasma 
PINCH level at 24h-post heading, but plasma PINCH at 0h- and 2h-post heading will 
remain undetectable, while there will be a significant increase in serum tau level at 
immediately post-heading (0h and 2h) but normalize to baseline level at 24h-post 
heading. 
After a concussion, a neural network is often disturbed due to axonal injury, 
observed via neuroimaging.34, 35 Also, an acute increase in plasma tau has been 
observed in concussed athletes.36 However, a half-life of tau protein in the blood is 
known to be short (~3h),37 thus at 24h-post heading tau level is expected to 
normalize to the pre-heading baseline level. Recent preliminary findings showed 
that neural disturbance was notable in football players at post-season compared 
with pre-season baseline, as well as rugby players after a single rugby game 
compared with pre-game level.38, 39 Although subconcussive impacts do not cause 
concussion-related symptoms,18 neuronal axons are evidently experiencing 
pathological stress.40 Repetitive subconcussive impacts likely to cause tau injury, 
and because PINCH continues to be over-expressed in the brain as long as tau is 
 
Table 1. Research studies examining the effect of an acute bout of soccer heading 
Author  
Subject 
Method 
Outcome 
Patukian et al. (2000) 
100 college athletes 
20 minutes of practice heading 
NSD in neuropsychological performance 
Broglio et al. (2004) 
40 college Athletes 
20 headers in 20 minutes 
NSD in postural control 
Magnus et al. (2004) 
10 college Athletes 
20 headers in 5 minutes 
NSD in balance 
Schmitt et al. (2004) 
31 college athletes 
18 headers in 40 minutes 
NSD in postural control, Symptoms  
increased (headache, vertigo, fatigue) 
Mussack et al. (2003) 
amateur athletes 
61 heading group 
58 active controls 
55 min of heading practice 
Exercise 
Significant increase in S-100B in heading versus active 
control group 
Stalnacke et al. (2004) 
44 professional 
athletes 
Soccer game 
Significant increase in S-100B, +Correlation between S-
100B and # of headers 
Otto et al. (2005) 
adult athletes 
Boxing  
Marathon Running 
Soccer Heading 
 (16 mph) 
Significant increase in S-100B 
Significant increase in S-100B 
NSD in S-100B 
Haran et al. (2013) 
16 college athletes 
Soccer Heading (25 mph) 
Significant change in postural control using virtual 
environment 24h post. 
Zetterberg 
et al. (2007) 
23 amateur soccer 
players 
10 Non-athletic 
subjects 
20 headings 
NSD in S100B between pre vs. post and heading vs. 
control group.   
Dorminy et al. (2015) 
16 college soccer 
players 
5 headings at 30, 40, 50 mph 
NSD in S100B nor concussion symptoms after soccer 
heading at any speed.  
Kawata et al. (2016) 
20 college soccer 
players 
10 headings at 25mph 
Significant decrease in ocular-motor function immediately 
after and 24h post heading. 
Hwang et al. (2016) 
20 college soccer 
players 
10 headings at 25mph 
Significant change in vestibular function at immediately 
after headings but NSD in 24h post-heading. 
Kaminski et al. (2007) 
71 college soccer 
players 
Pre-post Season 
NSD in balance, neurocognitive performance 
Kontos et al. (2011) 
63 youth soccer 
players 
Heading exposure groups 
High vs. Moderate vs. Low 
NSD in neurocognitive performance between groups 

damaged,21, 22 a fraction of PINCH will be detectable in the peripheral blood stream 
via glymphatic system or disrupted blood-brain barrier.37 
 
Aim 2. Determine acute effects of subconcussive head impact on dynamic gaze-
target synchronization in a controlled soccer heading model.  
Hypothesis: Repetitive subconcussive head impacts will significantly decrease saccadic 
velocity and smooth pursuit velocity gain, and increase gaze position error variability. 
Soccer heading model is a safe and reliable method to study mild head impacts, 
with average impact magnitude of 14 g.17, 33 To track a moving object, the brain 
must predict the trajectory of the moving target and maintain focus by concomitant 
adjustment of eye movement. We will employ the Eye-Sync system, which 
objectively measures important parameters, including smooth pursuit velocity gain, 
saccadic peak velocity, gaze position error variability.  
 
Aim 3. Inspect subconcussive effects on changes in neuronal activity during 
resting default mode. 
Hypothesis: Subconcussive impacts will induce a significant suppression of theta, alpha 
and beta waves (powers) in the frontal and occipital brain region during resting default 
mode, compared with baseline levels. 
Since subconcussive impacts induce diffuse force to the brain, rather than a focal 
injury like contusion,4 likely neuronal network is disrupted. Neuronal activity –delta, 
theta, alpha, beta powers– in the whole brain will be measured using 
electroencephalography (EEG) while resting.  
 
Aim 4. Determine whether subconcussive head impacts induce physiological 
effects in primary motor cortex. 
Hypothesis: Subconcussive impacts will significantly reduce primary motor cortex 
excitability compared with baseline levels.  
To better characterize neuronal physiology after subconcussive impacts, we will 
employ transcranial magnetic stimulation (TMS) to detect subclinical, physiological 
change in the primary motor cortex (M1). Consistent with established protocol,41 
we will target the M1 representation of a finger muscle at baseline and each post-
heading time point to compute the input/output (I/O) curve, which reflects M1 
excitability.  
 
3.0 
Inclusion/Exclusion Criteria 
 
Inclusion Criteria 
For Soccer Cohort 
1) being between 18 to 26 years of age  
2) an active member of a soccer team (i.e., collegiate, intramural, club, professional) 
3) at least 5 years of soccer heading experience. 
 
For non-Athletic Control Cohort 
1) Being between 18 to 26 years of age 
2) Have never played organized sports 
3) Have never been diagnosed with a concussion 
 
Exclusionary criteria 
For both Soccer and Non-Athletic Control cohorts  

1) any head, neck, or face injury in the 1 year prior to the study (e.g., concussion, eye 
injury);  
2) history of vestibular, ocular, or vision dysfunction (e.g., macular degeneration)  
3) currently taking any medications affecting balance (e.g., antibiotics) 
4) pregnancy  
5) HIV 
6) any neurological disorders (e.g., seizure disorders, closed head injuries with loss of 
consciousness greater than 15 minutes, CNS neoplasm, spinal cord injury/surgery, 
history of stroke) 
7) hypertension, cardiac arrhythmia, or pulmonary disease  
8) lower extremity injury that would prohibit normal walking 
9) metal implants in the head 
10) implantation of cochlear device, cardiac pacemaker, medical fusion device, 
intracardiac lines, or neurostimulator (e.g., DBS, epidural/subdural VNS) 
11) history of severe injury to the bones, joints, or muscles in either arm 
 
Session-specific exclusion criteria will include: 
12) slept less than 4 hours before the 1st and 2nd test day (verified by the TMS 
screening questionnaire) 
13) drank more than 3 alcoholic drinks or used recreational drugs 24 hours before the 
1st and 2nd test day 
14) drank more than 3 cups of coffee in an hour before test sessions 
15) glasses are prohibited (contact lens are okay) for safety purpose for the heading 
intervention 
 
4.0 
Enrollment/Randomization 
 
Potential participants will be recruited via listserv email to undergraduate students in the 
School of Public Health-Bloomington, and interested participants will contact (phone or 
email) and meet with the investigator to discuss the project and ask questions. The 
informed consent and Concussion History & Health History Questionnaire will be given to 
the potential candidates. Participants who meet the inclusion criteria and are free of 
exclusionary factors will advance to the testing procedures. 
 
We will use simple randomization method for group assignments. Up on inclusion of the 
participants, based on dice roll they will be randomly assigned into one of three groups: 
Heading group (number of dice 1 or 2), kicking group (number of dice 3 or 4), or standing 
group (number of dice 5 or 6). After assigned to groups, based on flipping a two-sided coin, 
participants will be further assigned into either TMS (head) or EEG (tail) experiments.   
 
Dr. Kawata (PI) will only know subjects’ group assignment and ensure blinded experiment 
and data processing. Dr. Kawata will randomly assign participants into three groups and 
perform soccer heading/kicking/standing protocol, and draw blood. Ocular-motor testing and 
data processing will be performed by Ms. Coon and blood biomarker experiments will be 
performed by Ms. Wirsching. The EEG experiment will be conducted by Mr. Innis and 
analyzed by Dr. Newman. The TMS experiment will be performed by Ms. Mirdamadi and 
analyzed by Dr. Block. As noted, statistical analyses of all the data will be performed by Dr. 
Chen. These process will ensure single blind method.  
 
5.0 
Study Procedures 

 
Research Design 
We will use a repeated measures design. The independent variable will be time (pre-
test, 0h post-test, 2h post-test, 24h post-test). Control groups (kicking and standing) will 
be used to analyze any effects from bodily damage, exercise, and normal changes in 
dependent variables over time. The dependent variables assessed over time will include 
symptom scores, ocular-motor function, blood biomarker concentration, brain waves, 
and cortical excitability. Impact kinematics will also be assessed during soccer heading.   
 
The study consists of 4 data collection time points in 2-day period. The 1st collection - 
pre-heading baseline-, followed by soccer heading intervention. The 2nd collection, 0h-
post-heading time point (immediately after headings). The 3rd collection, 2h-post-
heading time point (2h after headings). The 4th collection, 24h-post-heading time point 
(2nd day). This study design will enable to test outcome measures over 3 acute phases 
(Figure 5). The 1st day will last 4.5 hours and 2nd day will last 1.5 hours. Since we 
propose to include a total of 60 subjects in the study, we estimate data collection and 
primary 
analysis 
of 
60 
subjects 
will 
last 
approximately 
15 
months. 
 
In order to address the potential residual effects of previous soccer-related injury or 
head trauma, we will recruit additional 20 age- and gender-matched healthy-non-athletic 
controls, against subjects in the soccer heading group to validate whether baseline 
values of each parameter are comparable to a healthy non-athletic cohort. However, 
regardless of whether there will be a difference in baseline between soccer players and 
healthy-controls, the purpose of the current study is to determine acute subconcussive 
head impact effects in the athletic population. Thus, the changes between soccer 
players and healthy controls will not terminate the study.  
 
Soccer Heading Protocol 
When a subject who suffice inclusion criteria and free of exclusion criteria agree to 
participate in the study, he/she will be randomly assigned into one of three groups 
(heading, kicking-control, standing-control). A standardized and reliable soccer heading 
protocol will be used for the experiment. A triaxial accelerometer (Triax Technologies) 
embedded in a head-band pocket and positioned directly below the external occipital 
protuberance (inion) to monitor linear and rotational head accelerations. A JUGS soccer 
machine will be used to simulate a soccer throw-in with a standardized ball speed of 
25mph across all 3 groups. The ball speed is similar to when soccer players make a long 
throw-in from the sideline to mid-field. Soccer players frequently perform this maneuver 
during practice and game. Subjects will stand approximately 40ft away from the 
machine to perform either the heading, kicking, or standing (Fig 5 & 6). Dr. Kawata's 
previous laboratory at Temple University has routinely employed the protocol and 
average linear head acceleration yielded from the header is 14.5g in which many of 
subconcussion studies in football and ice hockey have set a threshold of impact 
recording as 16g, indicating that impacts induced from our protocol are below the 
minimum magnitude recorded in other contact sports. Participants in the heading group 
perform 10 standing headers with 1 header per minute, whereas kicking control group 
performs 10 kicks. Standing control group will remain static while a soccer ball passes 
over the head. The subjects in the heading and kicking groups will be instructed to 
direct the ball back toward the JUGS soccer machine in the air, while the subjects in the 
standing group will remain static. The kicking and standing control groups will aid to 

distinguish effects of subconcussive impact from exercise, bodily damage, and/or simply 
a daily variation of the outcome measures. The first phase of acute subconcussive 
effects will be measured at 0h-post heading, indicating immediate sensory perturbation 
and neuronal damage, followed by measurement of the second phase, 2h-post neural 
inflammatory response. Lastly, ocular-motor function coupled with neural activity at 24h-
post heading will be measured to assess the third phase of subconcussive effects. 
 
 
 
 
 
 
Procedure Outline 
1. Email will be distributed 
2. Interested participants will contact PI (KK) to discuss Project 
3. Participants meet with the PI to discuss the project and ask further questions. 
4. Participants take informed consent form with them and return with signature if they are 
willing to participate in the study 
 
a. Pre-test 
1. Symptom Assessment (Neurotrauma Lab; 5 mins, KK, SC, AW) 
(~40 mins if 
TMS) 
2. Ocular-motor assessment (Neurotrauma Lab; 10 mins, KK, SC) 
(~1h if EEG) 
3. Blood Draw (Exercise Biochemistry Lab; 5 mins, KK, SC, AW) 
 
4. TMS Assessment (Neuromotor Lab; 
15-20 mins, HB, JM) 
4. EEG Assessment (EEG lab; 30-40 
mins, SN, II) 
 
 
Intervention: Soccer heading, kicking, or standing (10 mins) 
 
 
b. 0h-Post-test 
1. Symptom Assessment (Neurotrauma Lab; 5 mins, KK, SC, AW) 
(~40 mins if 
TMS) 
2. Ocular-motor assessment (Neurotrauma Lab; 10 mins, KK, SC) 
(~1h if EEG) 
3. Blood Draw (Exercise Biochemistry Lab; 5 mins, KK, SC, AW) 
 
4. TMS Assessment (Neuromotor Lab; 
15-20 mins, HB, JM) 
4. EEG Assessment (EEG lab; 30-40 
mins, SN, II) 
 
 
 
c. 2h-Post-test 
1. Symptom Assessment (Neurotrauma Lab; 5 mins, KK, SC, AW) 
(~40 mins if 
TMS) 
2. Ocular-motor assessment (Neurotrauma Lab; 10 mins, KK, SC) 
(~1h if EEG) 
3. Blood Draw (Exercise Biochemistry Lab; 5 mins, KK, SC, AW) 
 
4. TMS Assessment (Neuromotor Lab; 
15-20 mins, HB, JM) 
4. EEG Assessment (EEG lab; 30-40 
mins, SN, II) 
 
 
 
1. 24h-Post-test 
1. Symptom Assessment (Neurotrauma Lab; 5 mins) 
(~40 mins if 
TMS) 
2. Ocular-motor assessment (Neurotrauma Lab; 10 mins) 
(~1h if EEG) 
3. Blood Draw (Exercise Biochemistry Lab; 5 mins) 
 
4. TMS Assessment (Neuromotor Lab; 
15-20 mins, HB, JM) 
4. EEG Assessment (EEG lab; 30-40 
mins, SN, II) 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
Symptom Checklist  
the  post-concussion  symptom  scale,  as a subset  of  
the  Sports  Concussion  Assessment 
Tool 3,3 will be used as a method of assessing the presence and severity of symptoms. 
This  paper-pencil  symptom  checklist  consists  of  22  symptoms  with  7-point  Likert  scale 
per  symptom  to  monitor  subject’s  well-being. Participants  will  be  
instructed  to  report 
their current symptoms  truthfully  by  circling  symptom  scores. Symptom  scores  will  be 
manually  transferred  into  an Excel  spreadsheet  for  the  future  analyses.  A  tester  or 
transferer will be blinded in regards to the group assignments and one’s performance in 
other test parameters. 
  
 
Blood Biomarker Assessment 
The  subject  will  be  asked  to  lay  on  supine  position  with  arm  relaxed  at  10-degree 
flexion.  Antecubital  vein  blood  draws  will  be  performed  each  test  session  to  help 
determine  serum  biomarker  concentrations.  A  certified  phlebotomist  will  thoroughly 
clean  the  antecubital  fossa  with an alcohol  swab  and  draw  4  ml  of  whole  blood  
into 
serum vacutainer tubes with a 21G butterfly needle. A Total of 16 ml blood for the study 
(4 ml x 4 time points). For non-athletic control subjects, 1 blood sample of 4 ml will be 
drawn. After the blood draw, the subject will use gauze to maintain direct pressure for 3 
mins  and  Band-Aid  will  be  provided.  The  whole  blood  will  be  centrifuged  at  3,000 
revolutions per minute for 20 minutes at 4°C after 40 mins of coagulation.  The serum 
will then be divided and transferred into 1 mL cryovials and flash frozen and stored at -
80°C in Exercise Biochemistry lab.  
 
Ocular-Motor Assessment 
0h-pre
ü Questionnaire
ü Ocular-motor
ü EEG
ü TMS
Blood Sample
Blood Sample
Blood Sample
STUDY DESIGN
Soccer 
Heading
Control 
(Standing)
Intervention
0h-post
ü Questionnaire
ü Ocular-motor
ü EEG
ü TMS
Control 
(Kicking)
3h-post
ü Questionnaire
ü Ocular-motor
ü EEG
ü TMS
24h-post
ü Questionnaire
ü Ocular-motor
ü EEG
ü TMS
Blood Sample
Fig 5. Study design. 
3x4 repeated 
measures design will 
be used to assess 
acute effect of 
subconcussive head 
impact. 
40ft (12m)
SUBJECT
BALL SPEED 
25 mph (11.2 m/s) 
JUGS MACHINE
Total 10 Headers: 10 –23 g per header 
Fig 6. Mild head impact 
intervention, previously used 
in our previous works (Hwang 
et al 33 and Kawata et al.17).  

The subject will then perform eye-movement task using the EYE-SYNC headset. This 
visual-tracking protocol has been replicated and validated in a number of concussion 
and sleep deprivation studies,42-47 however to our knowledge this study for the first time 
will unravel subconcussive effects. Prior to testing, a Snellen chart will be used to verify 
that the subject has a normal or corrected-to-normal vision (minimum 20/30). The 
subject will be seated in a normally lit room and stabilize the headset with two hands 
while the elbows placed on the desk. The visual stimulus will be presented using a 120-
Hz frame rate LCD screen in the headset and binocular eye movements will be tracked 
by a single camera secured in the headset. The test stimulus consists of a red circular 
target, 0.5° diameter in a visual angle with a 0.2° black dot in the center. The target 
moves in a circular clockwise trajectory of 10° radius at 0.4 Hz, with the target speed 
corresponding to 25°/s. The entire testing sequence will last approximately 3 mins 
consisted of a calibration and 2 consecutive test runs. Calibration of the eye position is 
conducted by having the subject fixate on a target presented at eight locations on the 
circular path of the test stimulus and one additional fixation point at the center of the 
circular path. The fixation target was presented at these nine locations in a randomized 
order. Each of the two test runs consists of 6 cycles of circular movement corresponding 
to 15 s in duration per test run. The subject will be instructed as “follow the movement 
of the target as closely as possible.” 
 
Transcranial Magnetic Stimulation Assessment 
After eye movement tasks, a subset of subjects (n=10) per group will participate in a 
transcranial magnetic stimulation (TMS) experiment, while the different subset of 
subjects (n=10) per group will participate in an electroencephalography experiment. 
These pilot studies are to assess subconcussive effects on neural activities. TMS will be 
used to assess physiological change in the primary motor cortex after subconcussive 
impacts. TMS works by electromagnetic induction: a current passed briefly through a coil 
of wire held over the scalp induces a magnetic field around the coil which, in turn, 
induces an electrical current in the underlying nerve cells on the surface of the brain. 
We will place electrodes in the hand to assess the cortical representation of an index 
finger muscle, first dorsal interosseous (FDI).  
 
During TMS application, both subject’s arms will be relaxed on a pillow. Subjects will 
wear goggles that are tracked by a Brainsight neuronaviation system, allowing us to 
register the subject’s head to the dimensions of a reference structural MRI. The hot spot 
for FDI will be determined at the beginning of each session and stored in Brainsight so 
that all stimulations within a session are applied at the same site. TMS above a certain 
threshold induces current in the cortex that activates neurons within the stimulated 
area. This results in a muscle twitch or motor evoked potential (MEP), measured with 
electromyography (EMG). Electrodes will be placed over the muscle belly and tendon, 
with a single common ground electrode over the ulnar styloid process of the target arm. 
EMG recording will be amplified, band-pass filtered (10-1000 Hz), and sampled at 5000 
Hz. MEP amplitude reflects the excitability of the stimulated area. The slope of the 
input/output (I/O) curve, computed from MEP amplitudes at a range of stimulation 
intensities, will be examined to determine the changes in corticospinal excitability at 
each time point. Including preparation steps, the TMS experiment will last approximately 
15 mins. 
  
 

Electroencephalography Assessment  
For EEG experiment, we will employ a standard 62-channel electro-cap with Ag/AgCl 
electrodes mounted to measure brain waves during default resting mode. To this end, 
the subject will be seated with eyes closed for 10 mins to ensure at least 2-min artifact 
free neural activity data.  The 62 channels will be collapsed into four topographical 
regions of interest (frontal, central, parietal, and occipital) in each hemisphere (left and 
right). The EEG data will be processed offline using EEGLAB 5.03 software. Including 
preparation steps, the EEG experiment will last 30-40 mins. 
 
 
 
6.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk 
to Participants or Others 
 
Reporting Adverse Events 
Keisuke Kawata, Ph.D. (primary investigator) will serve as the data safety monitor. The 
data safety monitor will monitor adverse event data, oversee procedures designed to 
protect the privacy of subjects, and coordinate the reporting of the outcome of any 
investigation of an adverse event. In the event of an adverse event, Dr. Kawata will 
report and cooperate with the IRB in any necessary investigation. 
 
Adverse Events Associated with Soccer Heading 
There is a risk of inducing symptoms such as headache, blurred vision, and 
disorientation during soccer heading testing, however ten soccer headers with mild head 
acceleration are commonly incorporated in soccer practice, and certainly soccer players 
utilize heading skill to direct a soccer ball for passing and scoring during practice and 
game. An average soccer player performs 500 headers over an entire season.30, 31 It is 
extremely unlikely to elicit concussion-like symptoms (headache, nausea, disorientation, 
blurred vision) from headings with mild head acceleration (Avg. 14.5g). The study 
protocol has been replicated in multiple laboratories, producing invaluable data to 
progress traumatic brain injury research. Moreover, our preliminary study in football 
players have shown that a player sustained 39 head hits with the total magnitude of 
1,200g during a single practice, but with no change in symptoms.18 In order to minimize 
the risk of adverse events from soccer heading, we will only include soccer players who 
regularly play soccer and perform soccer headings.  
 
 
 
Adverse Events Associated with Blood Draw 
Complications of antecubital venous blood draws include bruising, local infection, 
phlebitis and injury to structures near the cubital vein. All of these complications are 
uncommon. To minimize these risks, Keisuke Kawata, Ph.D., (a certified phlebotomist) 
will perform the blood draws using single-use needles, tube holders and test tubes. The 
skin of the antecubital region will be cleaned with alcohol to further reduce the risks of 
infections. Following each blood draw the participant will remain seated while 
maintaining direct pressure against the site of the needle insertion. After each blood 
draw, the participant will be given a Band-aid to cover the puncture site. When the 
participant returns for each test session the certified phlebotomist will check the 

puncture sites for any signs of complications (which would be referred for an additional 
medical examination).  
 
Following the 48 hour post-test, the participant will be reminded to contact the 
research staff if they have any pain or discomfort associated with the procedure. 
Pain and dizziness/fainting are other potential risks associated with antecubital venous 
blood draws. During the blood collection, the participants will be placed in a supine 
position. Prior to sitting up the certified phlebotomist will talk to the participant to 
confirm that the individual is comfortable. Once the participant is in a seated position 
they will sit for a few minutes to ensure that they are not feeling dizzy or faint. Ice will 
be offered at the end of each testing session to minimize any pain. 
 
 
Adverse Events Associated with Ocular-motor testing 
During performing ocular-motor tasks, participants may experience a transient headache 
and dizziness. Because the ocular-motor testing runs for 15 seconds and repeated twice, 
we believe that it is rare to elicit symptoms in the short period of testing. Based on the 
large-scale normative data (n=50,000) acquired by SynkThink Inc. among sports 
athletes and military servicemen, no one has claimed abnormal symptom due to the 
ocular-motor testing.   
 
 
Adverse Events Associated with Electroencephalography (EEG) 
There is minimal risk associated with EEG. An EEG measures the electrical activity in the 
brain (brain waves) using electrodes (small metal discs or sensors) placed on the head 
with gel. The test is non-invasive and no potential harm in neural structure nor 
metabolic balance, and the test does not hurt. The gel used to put the discs on the head 
is sometimes sticky and the discs may scratch the scalp. There may be a slight 
discomfort associated with sitting still in one position for 10-20 minutes. The likelihood 
of participant experiencing electric discharge from the EEG equipment is extremely 
small. No adverse events have been reported as a result of the EEG experiment. 
 
Adverse Events Associated with Transcranial Magnetic Stimulation (TMS) 
There is minimal risk associated with TMS.  In particular, we are using magnetic brain 
stimulation due to it being well-tolerated, versus direct electrical stimulation of the brain, 
which is very painful. TMS briefly activates nerve cells in a small area on the surface of 
the brain. The stimulator device uses the principle of ‘electromagnetic induction’ to 
produce a weak electric current on the surface of the brain. While the physical risk is 
almost none (assuming they have completed the TMS Checklist), there is the potential 
for the subject to have an adverse psychological reaction to the stimulation techniques. 
The possible side effects as it has been mentioned in literature are: seizure induction 
(rare), syncope (epiphenomenon), transient headache, transient neck pain, transient 
toothache, transient paresthesia, and transient hearing changes. Based on safety 
literatures on paired-pulse, there is no extra risk in paired pulse comparing to the single 
pulse. There is no reason to believe that single pulse TMS, itself, poses any hazard. 
Extensive use in Dr. Block’s previous laboratory at Johns Hopkins School of Medicine has 
not resulted in any difficulties with the device that could pose a hazard to participants. 
TMS does not appear to pose any hazard to the brain beyond that of electric stimulation, 
which has been in clinical use for decades.  The procedure appears to be safe and 
without any side effects.  Some of the original subjects have been stimulated many 

thousands of times. Some people may experience a mild headache from the stimulation. 
A headache, if it occurs, is usually mild and only lasts a few minutes beyond the end of 
the test. However, participants with a history of a migraine or other types of severe or 
frequent  
headaches  
will  
be 
excluded  
from  
the 
experiment. 
 
7.0 Study Withdrawal/Discontinuation 
 
Data collection will require subjects to participate in 4 test sessions. Subjects may 
withdraw from the study at any time. A subject may also be withdrawn from the research 
without his/her consent if, for example, he/she 1) comes to the 1st and/or 2nd day with 
intoxication, 2) sustained injury prior to the 1
st and 2nd day, 3) self-reported sleep 
deprivation for the 1st and/or 2nd day, 4) sustained a head injury prior to the 1st and 2nd 
day. If the subject completed for example entire 1st day procedure (3 test sessions) and 
unable to participate in the 2nd day (4th test session), the subject will be reimbursed 
hourly for the 1st day.  
 
For non-athletic control cohort, we will evaluate and account for the soccer players
' 
baseline, which may be influenced by a history of repetitive heading and sports 
participation. This group will participate in a single test session that lasts approximately 
in 1.5 hours. During the session, blood draw (4mL, about 1 teaspoon), measure eye 
movements using an ocular motor headset, and measure the brain activity using 
transcranial magnetic stimulation and electroencephalography. A subject may be 
terminated for the study participation if he/she 1) comes to the test session with 
intoxication, 2) sustained injury between screening and the day of test session, 3) self-
reported sleep deprivation on the day of test session.  
 
 
8.0 Statistical Considerations 
 
Power analysis 
According to our previous studies [Kawata et al. (2016)17 and 
Hwang  et  al.  (2016)33]  and  preliminary  findings  (Fig  4), the 
effect size is estimated to be 0.55 and the standard deviation 
is approximately 0.6. To achieve a power of 0.80 with p<0.05 
as significance level, 18.68 subjects per group are suggested 
for  the  studies (Formula  on  the  right).  Although  we  did  not 
have  any  drop-outs  in  aforementioned  previous  studies,  the  study  would  need  20 
subjects  per  group  to  be  able  to interpret  the  data  with  any  degree  of  confidence 
regarding  the  effects  of  subconcussive  head  
impacts  on  outcome  measures. Since  we 
are  recruiting  10  subjects  per  group  for  TMS  and  EEG  for  pilot  studies,  subsequent 
power analyses will be performed following data collection to determine whether we will 
need additional sample size or not.    
 
Data analysis 
Data  analysis  will  be  conducted  by  Dr.  Zhongxue  Chen,  associate  professor  of 
biostatistics  and  director  of  biostatistics  counseling  center.  To  maintain the purity  of 
statistical analyses, Dr. Chen will be blinded to group assignments by simply given the 
data  set  
indicating  Group  (A,  B,  C).  However,  for  our  statistical  model  of  
longitudinal 
assessment, time points (pre, 0h-post, 2h-post, 24h-post) will be revealed to Dr. Chen. 
n= 2(1.96 + 0.8416)2(0.6)
2
Z
a
Z
1-B
SD
(0.55)
2
Effect Size

Our primary interest is to identify the changes in biomarker expression, ocular-motor, 
and neural function after subconcussive head impacts in the heading group; hence, each 
parameter at post-heading time points (0h, 2h, and 24h) will be compared with pre-
heading baseline. Each time point of the control groups (kicking and standing) will aid to 
interpret whether or not changes in the heading group are due to subconcussive impact, 
exercise, bodily hit, or simply a daily variation. To this end, a series of mixed effects 
regression models (MRM) with random intercept will be used for all aims.48 The first 
MRM will focus on the within-group pattern of changes in biomarker expressions (PINCH 
and Tau),  ocular-motor performance (smooth pursuit velocity gain, phase error, and 
gaze position error), neural activity (delta, theta, alpha, and beta powers), and motor 
cortex excitability (I/O curve) in 3 groups across the duration of the study – 4 time points. 
Next MRM will focus on between-group analyses on the variables (heading, kicking, 
standing) against changes in the biomarker levels, ocular-motor function, and neural 
function. Predictor variables include time, group, and time by group interaction. All 
analyses will be conducted with SAS and significance will be set at p < 0.05. 
 
 
Study Endpoint 
 
Throughout the study, we set the study endpoint based on subjects’ well-being 
measured via symptom checklist or verbal claim to researchers. Because the mild nature 
of head impact, we expect to observe a subtle change in our sensitive outcome variables 
after soccer headers. To date, there is virtually no scientific evidence available to set 
study endpoints using ocular-motor, blood biomarker, and neuronal activity, we will guide 
the study based on symptoms, meaning that if a subject experiences any concussion-
related symptoms following blood draw, soccer heading, ocular-motor testing, EEG, and 
TMS, his or her participation will be terminated. 
 
 
Symptom 
When a subject experiences any symptoms related to concussion (headache, nausea, 
blurred vision, disorientation, fogginess, sensitivity to light and noise, etc.), his/her 
participation will be terminated. However, there has been no participant thus far reported 
concussion-related symptoms due to soccer heading, blood draw, nor ocular-motor 
performance. 
 
Blood Biomarker 
 
Presently, there are no biomarker concentration levels that can serve as a cut-off point 
to diagnose concussion or subconcussion, while in more severe traumatic brain injury, 
astrocyte-derived biomarker (S100B and Glial Fibrillary Acidic Protein) have known to be 
useful in prognosis of brain injury and its outcome.49-51 The proposed study tests 
neuron-enriched blood biomarkers (PINCH and tau proteins), where theoretically 
detecting these markers in the blood indicates neuronal damage in the brain. Our 
preliminary work on PINCH (Fig 4) illustrates that season-long exposure to 
subconcussive head impacts in football players induced increases in PINCH protein, 
while PINCH remains undetectable in 14 out of 16 players. However, there is no 
scientific evidence thus far demonstrating the association of PINCH/tau proteins in the 
blood and brain damage. The study design we employ allows us to test the ambiguous 
notion whether head impacts may cause the increase in PINCH and/or tau in the blood 
and possible association with neural activity measured via TMS and EEG.  
 

Neuronal Activities 
 
Altered neuronal functions will be measured via TMS and EEG. As noted in the 
hypotheses under Specific Aims, following subconcussive head impacts, we expect to 
observe a transient suppression of theta, alpha and beta waves (powers) in the frontal 
and occipital brain region measured via EEG, indicating temporary neuronal perturbation 
due to head impacts. Similarly, using TMS, we will target the M1 representation of a 
finger muscle at baseline and each post-heading time point to compute the input/output 
(I/O) curve, which reflects M1 excitability. If the subject experiences any adverse 
symptoms which are similar to concussion-related symptoms (headache, nausea, 
blurred vision, disorientation, fogginess, sensitivity to light and noise, etc.), he or she will 
terminate the study participation.  
 
 
 
 
9.0 
Privacy/Confidentiality Issues 
 
All participant information, and even the fact that an individual is in the study, is 
considered confidential. Confidentiality will be assured in this study through several 
mechanisms. During interviews and treatments, the investigators and study coordinator 
will ensure physical privacy by conducting interviews in a closed room. Subjects will be 
assigned a subject number to help make data anonymous. The participant’s Protected 
Health Information will be used for research purposes only.  No names will accompany 
any data that is used for publication. To reduce the risk of confidentiality loss, electric 
data collected during the study will be stored on the university server and data collection 
sheets will be stored in a locked file cabinet in a locked room. The data will be stored 
indefinitely for data quality purpose for potential investigation after publishing the data.  
Only study personnel will have access to the data. All members of the research team are 
certified through the CITI program.  Individual subject results will not be shared with 
the participants or their agents. Data analysis and publication will not include any 
identifying information. 
 
Data Management 
 
For symptoms, blood biomarker, and ocular-motor function, we will utilize spreadsheet 
to organize data and store in the IU Box account. Microsoft excel does not have range-
checks system, however when we run statistical analysis using Statistical Analysis 
System (SAS), which can identify outlier and appropriate range for acquired data.   
 
Symptom 
Symptom scores will be manually transferred into an Excel spreadsheet for the future 
analyses. A tester or transferer will be blinded in regards to the group assignments and 
one’s performance in other test parameters. The Excel spreadsheet will be saved in the 
university secured Box account, which will be backed up weekly. 
 
Ocular-motor function 
The eye-movement data acquired via the EYE-SYNC headset will be automatically saved 
in an Excel spreadsheet. The Excel spreadsheet will be saved in the university secured 
Box account, which will be backed up weekly. 
 

Blood Biomarker 
Biomarker expressions will be measured via Simoa HD-1 Analyzer and the data will be 
transferred into an Excel spreadsheet by the tester. 
 
EEG    
The EEG data will be processed offline using EEGLAB 5.03 software, which has a built-
in range-check system. The data will be backed up weekly. 
 
TMS 
The TMS data will be captured and processed using Brainsight software (Rogue 
Research), where outliers can be identified by a built-in range-check system.  
 
 
10.0 Follow-up and Record Retention 
 
Since we propose to include a total of 80 subjects in the study, we estimate data 
collection and primary analysis of 80 subjects will last approximately 15 months. The 
electric data collected during the study will be stored on the university server, and data 
collection sheets will be stored in a locked file cabinet in a locked room. The data will be 
stored indefinitely for data quality purpose for potential investigation after publishing the 
data.  
 
 
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