Placebo Controlled Phase II Clinical Trial: Safety and Efficacy of Combining Intranasal Insulin & Acute Exercise
Phase: PHASE2
Conditions: Insulin, Exercise
Sponsor: Michigan State University

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Study Protocol and Statistical Analysis Plan 
For Research Project: 
“Safety and Efficacy of Combining Intranasal Insulin & Acute Exercise” 
 
clinicaltrials.gov # NCT04292535 
 
conducted by Matthew. B. Pontifex, Ph.D. 
 
March 19, 2021 
 
 
 

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“Safety and Efficacy of Combining Intranasal Insulin & Acute Exercise” 
 
 
Abstract: To determine if physical activity engagement alters the dose-response profile 
and safety of administration of insulin into the intranasal mucosa. Using a randomized 
placebo controlled double-blind pre-posttest design, participants were randomly assigned 
to receive a dose of either 0, 20, 40, 60, 80, 100, or 120 IU of NovoLog insulin aspart prior 
to being randomized into participating in a 20 minute session of either moderate-intensity 
aerobic exercise or a passive control condition. The safety of the protocol was assessed 
using a symptom questionnaire assessing common symptoms of altered blood glucose 
and common side effects of intranasal insulin. The efficacy of the intranasal insulin for 
inducing alterations in cognition was assessed using both behavioral and neuroelectric 
measures.  
 
 
Aims:  
1) To provide a preliminary assessment of the safety profile of combining intranasal 
insulin with exercise; to ensure that undue risks are not incurred through 
combination therapeutic approaches. 
 
2) To provide a preliminary assessment of the efficacy of combining intranasal insulin 
with exercise for enhancing inhibitory aspects of cognitive control and sustained 
attention alongside neuroelectric indices of attention. 
 
 
Design: Double blind, placebo controlled Phase 2 clinical trial using serial stratification 
randomization accounting for biological sex. Participants were first allocated to a dose of 
intranasal insulin (0 [placebo], 20, 40, 60, 80, 100, 120 IU), then allocated to either 
exercise or control activity. 
 
 
Study Population: Given the potential risk of combining exercise with intranasal insulin 
the present investigation utilized a sample of healthy, non-diabetic, fasted college-aged 
adults to limit the potential for a serious adverse event. The sample consisted of college 
aged young adults from the mid-Michigan, USA area.  
 
 
Eligibility Criteria: 
Criteria for inclusion: All individuals that agreed to participate were selected on a first 
come, first serve basis. No individual was turned down due to sex, race, or ethnicity. 
The following inclusion criteria exist for all participants: 
a. Participants must be over the age of 18. 
b. Participants must have normal or corrected-to-normal vision in order to complete 
the cognitive task. 
 
Exclusion criteria: The following exclusion criteria exist for all participants: 

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a. Lack of consent. 
b. Presence of any major neurological health issues, brain trauma, or concussion 
with loss of consciousness assessed through a health history and demographics 
questionnaire. 
c. Type I or Type II Diabetes. 
d. Self-reported pregnancy. 
e. Currently has any type of inflammation or blockage of the nasal passageways (i.e. 
allergies or a cold affecting the sinuses). 
f. Inability to engage in treadmill-based exercise. 
 
 
Recruitment: Participants were recruited from the mid-Michigan community population 
via flyers and list-serve announcements.  
 
 
Protocol: This investigation was approved by the Michigan State University Human 
Research Protection Program. 
 
Screening Day (day 1): On the screening day of the testing protocol, following 
consenting to participate in this investigation; participants completed a brief health and 
history demographics questionnaire, a physical activity readiness questionnaire (Thomas 
et al., 1992), a medical screening questionnaire, the Wechsler Abbreviated Scale of 
Intelligence – 2nd Edition (WASI-II; (Wechsler, 2011), and a test of maximal oxygen 
uptake (VO2max; using the same protocols as reported in (Chandler et al., 2019; Pontifex 
et al., 2016)). The medical screening questionnaire ensured that all participants were free 
of medication for high blood pressure, cholesterol, asthma, heart issues, 
depression/anxiety; and were not currently on any beta-blockers, sulfonamide antibiotics, 
corticosteroids, protease inhibitors, or Clonidine.  
 
Participants who were eligible to continue in the study were then randomly assigned 
using a serial stratification approach accounting for biological sex to either an exercise 
[active experimental group] condition or sitting [control experimental group] condition. 
Within each condition; participants were subsequently randomly assigned using a serial 
stratification approach accounting for biological sex to receive a dose of either 0, 20, 40, 
60, 80, 100, or 120 IU of fast acting insulin (Novolog Insulin Aspart 100 mg/mL) 
administered into the intranasal mucosa. Participants were instructed to refrain from 
exercise or caffeine intake, and too fast for at least 3 hours prior to participating in the 
second day of the testing protocol.  
 
 
 

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Assessment day (day 2): See Figure 1 for an overview of the assessment activities. 
 
Figure 1. Diagram of experimental protocol. 
 
Upon arriving to start the second day of testing, participants’ blood glucose level was 
assessed via a glucometer (CVS Health Advanced Blood Glucose Meter, United States) 
to ensure that their blood glucose fell within the 70-115 mg/dL guideline to exercise safely. 
Following this, participants were outfitted with a polar heart rate monitor (Model H7, Polar 
Electro, Finland) to assess heart rate during the experimental condition and then were 
prepared for neuroelectric testing. Participants were subsequently seated in a sound-
attenuated testing chamber approximately 1 m away from the computer monitor where 
they completed tests of inhibitory control and sustained attention while behavioral and 
neuroelectric measures were assessed. Once the pretest cognitive assessment was 
completed, participants were administered solution into their intranasal mucosa. 
 
Using 
a 
double-blind, 
placebo-
controlled approach; identical vials of saline 
solution and Novolog insulin aspart 100 
mg/mL were obtained differentiated only by 
the color of the stopper top, and labeled as 
investigational drug A and investigational 
drug B. All participants were administered 6 
doses of 0.2 mL of solution via the LMA 
MAD Nasal Intranasal Mucosal Atomization 
device   into alternating nostrils with a total 
administration volume of 1.2 mL. The dose 
of intranasal insulin was controlled by 
varying the number of doses which 
contained saline relative to the doses which 
contained Novolog insulin aspart 100 
mg/mL (see Figure 2). The experimenters 
were aware of the number of doses of 
investigational drug A and investigational 
drug B administered; but were unaware 
which 
solution 
or 
stopper 
top 
was 
associated with insulin or saline. Following 
administration of the solution, participants 
completed the sitting/exercise experimental 
conditions.  
 
Figure 2. Illustration of the intranasal insulin 
dosing protocol. 

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Active Comparator: Passive Control. The sitting experimental condition consisted of a 
20 min duration where participants sat in a chair (HR = 73.2 bpm [95% CI: 70.3 to 76.2]).  
 
Intervention. The exercise experimental condition consisted of similar 20 min duration 
of exercise on a motor-driven treadmill at an intensity between 60 and 65% of maximum 
heart rate achieved during the test of maximal oxygen uptake (HR = 118.1 bpm [95% CI: 
116.2 to 120.1]).  
 
During both the sitting and exercise experimental conditions, participants watched an 
emotionally neutral video (minutes 65-85 from Wonders of the Universe (Wonders of the 
Universe, 2011)). Midway through the sitting and exercise experimental conditions and 
again 5 minutes following the completion of the experimental conditions, peripheral blood 
glucose was assessed via a glucometer. If peripheral blood glucose fell below 70 mg/dL, 
testing was immediately discontinued for all measures. Following the peripheral blood 
glucose assessment, participants again completed the tests of inhibitory control and 
sustained attention while behavioral and neuroelectric measures were assessed. Finally, 
upon completion of the cognitive assessments, participants completed a symptom survey 
which assessed potential side effects of intranasal insulin that participants might have 
experienced at any time during the experimental protocol and if participants were still 
experiencing those symptoms.  
 
 
Measures:  
 
Safety of Intranasal Insulin: The safety of intranasal insulin in combination with 
exercise was assessed by examining the incidence of participants reporting potential side 
effects of intranasal insulin (Schmid et al., 2018) at any time during the experimental 
protocol and the persistence of those symptoms. Additionally, the extent to which 
peripheral blood glucose was altered was also assessed via glucometer at the beginning 
of the session, during the middle of the exercise/sitting condition, and following the 
exercise/sitting condition 
 
Inhibitory Control Task: Inhibitory control was assessed using a letter version of the 
Eriksen flanker task (Eriksen & Eriksen, 1974; McGowan et al., 2019), which is classified 
as a behavioral response selection construct of the inhibition/suppression focus and the 
performance monitoring focus of cognitive control according to the NIMH Research 
Domain Criteria (RDoC) classification system. Participants were instructed to attend to 
and to respond as accurately as possible to a centrally presented stimulus nested amid 
either congruous (‘MMMMM’) or incongruous (‘NNMNN’) flanking stimuli. Participants 
completed 80 practice trials at pretest followed by 160 trials grouped into two blocks of 
80 trials, each consisting of equiprobable congruency and directionality; at each 
assessment period (160 trials pre-experimental condition and 160 trials post-
experimental condition). For each block of trials, participants were presented with 
perceptually similar letter pairs (e.g., pretest block 1: M – N, pretest block 2: E – F, posttest 
block 1: I – T, posttest block 2: U – V) and were instructed to respond by pressing the 
button assigned to the letter presented in the middle of the flanking letters.  

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To ensure a high degree of task difficulty, at the midpoint of each block participants 
were given instructions to reverse the button-letter assignments (e.g., left button press for 
“M” through the first 40 trials and then right button press for “M” through the last 40 trials). 
Flanking letters were presented 300 ms prior to the onset of the target letter, and all five 
letters remained on the screen for a subsequent 100 ms (for a total stimulus duration of 
400 ms) with a response window of 1000 ms and a variable inter-trial interval of 2300, 
2400, 2500, 2600, or 2700 ms. All stimuli were 1.5 cm tall white block letters presented 
focally on a black background. Stimulus presentation and timing were controlled using 
PsychoPy 1.86 (Peirce, 2009). Reaction time was quantified within each congruency as 
the mean speed of responding following the onset of the stimulus only for correct trials.  
Response accuracy was quantified within each congruency as the proportion of correct 
responses relative to the number of trials administered.  
 
Sustained Attention Task: Sustained attention was assessed using the rapid visual 
information processing task (Chandler et al., 2020; Neale et al., 2015) which is classified 
as a behavioral construct of attention according to the NIMH Research Domain Criteria 
(RDoC) classification system. Participants were presented with a series of single digits 
(1-9) in a box in the center of the screen at a rate of 100 digits/min and were instructed to 
make a button response with their right thumb as soon as they detected any of the three 
target sequences: ‘2-4-6,’ ‘3-5-7,’ or ‘4-6-8.’ To minimize working memory load, the three 
target sequences were presented on the bottom of the screen throughout the duration of 
the task. At pretest, participants completed a 1 min practice period prior to beginning the 
test trials which contained a series of 402 digits with 64 target sequences embedded. At 
posttest, participants completed another series of 402 digits with 64 target sequences 
embedded. Reaction time was quantified as the mean speed of responding following the 
presentation of the final digit of the target sequences only for correct sequence 
identifications. Response accuracy was quantified as the proportion of responses that 
correctly coincided with the 64 target sequences presented. 
 
Neuroelectric Indices of Attention: During completion of the inhibitory control and 
sustained attention tasks, EEG activity was recorded from 32 electrode sites (Fpz, Fz, 
FCz, Cz, CPz, Pz, POz, Oz, F7/3/4/8, FT7/8, FC3/4, T7/8, C3/4, M1/2, CP3/4, TP7/8, 
P7/3/4/8, PO5/6) arranged in an extended montage based on the International 10-10 
system (Chatrian, Lettich, & Nelson, 1985) using a Neuroscan Quik-Cap (Compumedics, 
Inc., Charlotte, NC). Recordings were referenced to averaged mastoids (M1, M2), with 
AFz serving as the ground electrode. In addition, electrodes were placed above and 
below the left orbit and on the outer canthus of both eyes to monitor electrooculographic 
(EOG) activity with a bipolar recording. Continuous data was digitized at a sampling rate 
of 2048 Hz and amplified 500 times with a DC to 70Hz filter using a Neuroscan Grael 
amplifier. The EEG data were then imported into EEGLAB (Delorme & Makeig, 2004), 
downsampled to 1024 Hz and prepared for temporal ICA decomposition. Data more than 
2 s prior to the first event marker and 2 s after the final event marker were removed to 
restrict computation of ICA components to task-related activity. The continuous data was 
filtered using a 0.05 Hz high-pass 2nd order Butterworth IIR filter to remove slow drifts 
(Pontifex, Gwizdala, et al., 2017), and the mastoids electrodes were removed prior to ICA 

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decomposition. ICA decomposition was performed using the extended infomax algorithm 
to extract sub-Gaussian components using the default settings called in the MATLAB 
implementation of this function in EEGLAB with the block size heuristic 
(floor[sqrt(EEG.pnts/3)]) drawn from MNE-Python (Gramfort et al., 2013). Following the 
ICA decomposition, the eyeblink artifact components were identified using the 
icablinkmetrics function (Pontifex, Miskovic, et al., 2017) and the EEG data was 
reconstructed without the eyeblink artifact. 
 
Following removal of the eye blink components, stimulus-locked epochs were created 
for correct trials from -500 to 1,500 ms around the stimulus, baseline corrected using the 
-100 to 0 ms pre-stimulus period, and filtered using a zero phase shift low-pass filter at 
30 Hz. Trials with artifact exceeding ±100 μV were rejected. To ensure the integrity of the 
signal, stimulus-locked epochs were visually inspected blind to the experimental condition 
and dose prior to computing mean waveforms. Following visual inspection, the mean 
number of trials included in the waveforms was 112.0 [95% CI: 107.2 to 116.7] trials 
stimulus-locked to the congruent and incongruent trials (separately) of the Inhibitory 
Control task and 41.6 [95% CI: 40.1 to 43.2] trials stimulus-locked to the final stimulus of 
the target sequences of the Sustained Attention task.  
 
Attention was indexed by the P3 ERP component. Attentional engagement was 
evaluated as the mean amplitude within a 50 ms interval surrounding the largest positive 
going peak within a 275 to 700 ms latency window following stimulus onset for the flanker 
task, and a 275 to 600 ms window for the RVIP task (Chandler et al., 2020; McGowan et 
al., 2019; Pontifex et al., 2015). Attentional processing speed was evaluated using ERP 
latency which was was quantified as the time at which maximum peak amplitude 
occurred.  Given the well-established nature of the P3 ERP component, analyses were 
conducted using a nine-channel region-of-interest approach centering around the 
topographic maxima of the P3 (i.e., the CP3/Z/4, P3/Z/4, PO5/Z/6 electrodes). 
 
 
 
 

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Aims Assessment:  
1) To provide a preliminary assessment of the safety profile of combining intranasal 
insulin with exercise; to ensure that undue risks are not incurred through 
combination therapeutic approaches. 
 
As the present investigation was a Phase II Clinical Trial, the research design is 
not sufficiently powered for hypothesis testing. Accordingly, this aim was satisfied 
by: 
 
A) Examining the frequency of participants reporting potential side effects of 
intranasal insulin at any time during the experimental protocol.  
 
B) Examining the frequency of participants reporting potential side effects of 
intranasal insulin that persisted at the end of the experimental protocol. 
 
C) Examining the frequency of study discontinuations due to violations of the 
safety thresholds (e.g., blood glucose outside the range of 70 to 150 mg/dL, 
feeling dizzy, or unable to safety continue the experimental protocol). 
 
2) To provide a preliminary assessment of the efficacy of combining intranasal insulin 
with exercise for enhancing inhibitory aspects of cognitive control and sustained 
attention alongside neuroelectric indices of attention. 
 
As the present investigation was a Phase II Clinical Trial, the research design is 
not sufficiently powered for hypothesis testing. Accordingly, this aim was satisfied 
by computing the within subject effect size for each participant as the standardized 
change relative to the pretest assessment using the within-subject (drm) variance 
correction for Cohen’s d (Lakens, 2013). To ensure the integrity of the effect size 
estimates, within-subject effect sizes exceeding 3 times the interquartile range 
were identified as outliers and removed from analysis. For each measure, within 
each experimental condition, the effect of intranasal insulin was corrected by 
subtracting the effect size observed for the 0 IU placebo group to isolate the effects 
of intranasal insulin. Preliminary assessments of the efficacy were thus quantified 
using: 
 
A) Effect size estimates with 95% confidence intervals for both control and 
exercise conditions collapsed across congruencies of the Flanker task to 
quantify the effects on Behavioral Indices of Inhibitory Control. Reaction 
time effects and response accuracy effects were separately extracted. 
 
B) Effect size estimates with 95% confidence intervals for both control and 
exercise conditions in response to the target trial of the Rapid Visual 
Information Processing task to quantify the effects on Behavioral Indices of 
Sustained Attention. Reaction time effects and response accuracy effects 
were separately extracted. 
 

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C) Effect size estimates with 95% confidence intervals for both control and 
exercise conditions for P3 amplitude at the nine-channel region-of-interest to 
quantify the effects on Neuroelectric Indices of Attentional Engagement. 
Effects collapsed across congruencies of the Flanker task and effects for the 
target trial of the Rapid Visual Information Processing task were separately 
extracted. 
 
D) Effect size estimates with 95% confidence intervals for both control and 
exercise conditions for P3 latency at the nine-channel region-of-interest to 
quantify the effects on Neuroelectric Indices of Attentional Processing 
Speed. Effects collapsed across congruencies of the Flanker task and effects 
for the target trial of the Rapid Visual Information Processing task were 
separately extracted. 
 
 
 

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