Acute Intermittent Hypoxia and Breathing in Neuromuscular Disease
Phase: NA
Conditions: Amyotrophic Lateral Sclerosis (ALS), Neuromuscular Diseases
Sponsor: University of Florida

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Acute Intermittent Hypoxia and Breathing in Neuromuscular Disease (AIH in ALS) 
 
 
NCT03645031 
 
 
November 20, 2020 
 

Title 
Acute Intermittent Hypoxia and Breathing in Neuromuscular Disease 
 
Investigators 
Barbara Smith, PI 
James Wymer Co-I 
Gordon Mitchell, Co-I 
Elaheh Sajjadi, Co-I 
Jennifer Steshyn, Coordinator 
Jessica Ehrbar, Coordinator 
 
Abstract 
Acute intermittent hypoxia (AIH) consists of alternating periods of breathing mildly hypoxic 
and normoxic air, and is a well-characterized stimulus to serotonin-dependent motor 
plasticity (1, 2). More than two decades of research from several independent laboratories 
indicates AIH elicits meaningful respiratory and non-respiratory motor recovery in both 
animal and human models of health and neurological injury, particularly chronic 
incomplete spinal cord injuries (3-7). Of particular importance, the “low dose” of 
intermittent hypoxia represented by AIH is well below that encountered with sleep apnea, 
and elicits functional benefits in diverse physiological systems without detectable 
pathology (8, 9). 
Our group is interested in the therapeutic potential of AIH for individuals with 
neuromuscular disease. In preliminary studies on rodent models of amyotrophic lateral 
sclerosis (ALS), brief AIH exposures restore lost respiratory function. Based on the 
efficacy of AIH in ALS rats (10, 11), and the success in translating this novel therapy to 
humans with chronic neurological impairment, the potential benefits of AIH in ALS 
patients warrant careful investigation. Most ALS patients survive less than 5 years after 
diagnosis, and the main cause of death is respiratory failure (12, 13). Few options are 
available to preserve breathing other than mechanical ventilation, which diminishes the 
quality of life to an extent that it is often rejected by ALS patients. Any treatment options 
to preserve/prolong independent breathing ability will greatly enhance the quality (and 
duration) of life in those suffering from ALS. Here, we seek proof of concept data that AIH 
may be a simple and effective therapeutic option to preserve independent breathing ability 
in ALS. 
The fundamental hypothesis guiding this proposal is that even a single AIH 
trial improves respiratory (and non-respiratory) motor function in ALS patients. We 
propose to evaluate respiratory and limb strength, minute ventilation and respiratory 
muscle EMG activity in ALS patients versus healthy age and sex-matched controls in 
response to AIH and sham AIH (normoxia) interventions. We will investigate the ability of 
a single AIH trial to increase voluntary respiratory motor function (Aim 1). An additional, 
exploratory aim tests the hypothesis that ALS patients utilize distinct patterns of 
respiratory muscle recruitment versus healthy controls, and that these patterns are 
partially normalized by AIH (Aim 2). Both aims are designed to collect preliminary safety 
and feasibility data as we develop methods that improve our ability to detect and treat 
respiratory impairment in ALS. 
  

Background 
Acute intermittent hypoxia (AIH) consists of short, repeated presentations of reduced 
oxygen in the inspired gas (~9-11% oxygen, ~1 – 1.5 minutes), interspersed with normal 
oxygen periods (~1-1.5 min, inspired O2=21%). AIH induced motor plasticity was first 
described more than two decades ago as a persistent enhancement of phrenic and 
hypoglossal neural activity lasting hours after AIH had ended. (14, 15) Subsequent 
research demonstrated that AIH preconditioning causes cumulative effects, and 
enhances the extent of motor facilitation induced by a single AIH presentation (16, 17). 
These observations triggered investigations concerning the potential of repeated AIH as 
a treatment to preserve and/or restore breathing ability with neural injury or disease (3, 
9). 
More than two decades of research on animal models have produced a detailed 
understanding of mechanisms giving rise to AIH-induced respiratory motor plasticity (3, 
4, 6, 18, 19). Briefly, each hypoxic episode activates ventilatory chemoreflexes initiated 
by carotid body chemoreceptors. Carotid body sensory activity stimulates medullary 
respiratory neurons that increase breathing (1, 2) and raphe neurons that contain the 
neuromodulator, serotonin. During each hypoxic episode in the AIH protocol, this neural 
network is repeatedly activated. The episodic release of serotonin near respiratory motor 
neurons is at the heart of AIH-induced plasticity versus low oxygen per se (4). Episodic 
activation of serotonin receptors on respiratory motor neurons initiates intracellular 
signaling cascades that trigger new brain-derived neurotrophic factor (BDNF) protein 
synthesis, strengthening synapses from medullary respiratory neurons that drive 
breathing (1, 6). Similar mechanisms occur in both respiratory and non-respiratory motor 
neurons as confirmed in numerous studies in both normal (3, 20, 21) and injury/disease 
rodent models (3, 22). When AIH is repeated daily for many days-to-weeks, the 
magnitude and duration of plasticity is amplified (22). 
The dose of intermittent hypoxia is critical in determining the direction and extent 
of plasticity, and the potential for adverse events (8). For example, high doses of 
intermittent hypoxia simulating obstructive sleep apnea [80 or more episodes per 
day/night; severe hypoxemia within each episode] elicit pathology, including systemic 
hypertension, neuroinflammation, and cognitive impairment (23, 24). On the other hand, 
respiratory motor plasticity is undermined by severe intermittent hypoxia protocols due to 
neuroinflammation (25). In striking contrast, repeated, low dose AIH [<15 episodes per 
day; modest hypoxemia per episode] elicits neuroplasticity without detectable pathology 
(26-31). To the contrary, repetitive low dose AIH exerts beneficial effects in multiple organ 
systems by independent research groups (for review see: (8)).  
Here, we propose to study mechanisms of respiratory plasticity associated with a 
single presentation of mild AIH. AIH is simple to apply and well tolerated by most 
individuals. In eight studies on humans with chronic spinal cord injury published to date, 
an AIH protocol similar to that proposed here has not been associated with any adverse 
events (5, 7, 31-35). Several other completed, but not yet published studies from multiple 
laboratories increase our collective experience with (and confidence in) this protocol 
(Christensen et al., submitted; Sandhu et al., in preparation; Ahmed et al., in preparation). 
All available evidence is consistent with the conclusion that AIH protocols similar to that 
proposed here are safe in humans, although we do not yet have experience with ALS 

patients. 
 
Accumulating evidence demonstrates that a similar AIH protocol to that proposed 
here induces respiratory motor plasticity in humans. For example, in healthy sleeping 
adults, tidal volume and ventilation acutely increase during each hypoxic episode, and 
remain above baseline >20 minutes post-AIH (36). Sustained motor facilitation is also 
observed in genioglossus EMG activity, presumably stabilizing the upper airway (37). 
Using a slightly different AIH protocol (with a background of mild hypercapnia), prolonged 
increases in tidal volume (and ventilation) are observed in awake humans with chronic 
spinal injury (34). In adult patients with chronic incomplete spinal injury, a single, 30-
minute AIH session (15, 1 min episodes) elicited >80% gain in ankle torque generation 
and EMG burst amplitude for up to 4 hours post-AIH (7). AIH-induced gains in limb muscle 
torque correlate with enhanced motor recruitment (5). Here, we propose to study 
effects of a similar, single AIH session in normal subjects and ALS patients to gain 
proof of principle that AIH elicits plasticity and meaningful functional benefits in 
this patient population. Although repeated, daily AIH exposure for 5 days extend the 
duration of motor enhancement to weeks (35), we do not yet feel justified in exposing ALS 
patients to such prolonged protocols pending the outcome of these initial experiments.  
Based on abundant experience in humans, the AIH protocol proposed here is 
noninvasive, easy to administer and, importantly, has no history of adverse effects. 
Further, when people with chronic spinal cord injury are asked which day they had 
received AIH versus sham normoxia treatment, they are unable to tell (their guess has a 
50/50% chance of being correct); thus, AIH at these levels is not stressful (indeed is barely 
detectable) for those with neurologic injury. The most notable discomfort reported during 
an AIH protocol is wearing the respiratory mask. Collectively, evidence from both animal 
models and humans with neurologic injury support the case that AIH is a safe, simple 
(non-invasive) and effective means of improving both respiratory and non-respiratory 
motor function, and that it has the potential to at least transiently improve motor function 
in diverse clinical disorders that compromise movement (9). 
Early results (n=6) indicate respiratory motor gains in some, but not all subjects 
following AIH. While the primary means of AIH motor facilitation occurs via serotonergic 
signaling, AIH motor facilitation can occur through an alternative, adenosine-mediated 
signaling pathway (38, 39). The serotonergic and adenosine pathways compete for 
dominance via cross-inhibition, and when activated equally, they cancel one another (19, 
40). However, recent basic science data from our group indicates that pre-treatment with 
an adenosine receptor antagonist can restore AIH-induced respiratory motor function 
(41). Aim 3 is an optional/exploratory aim evaluating whether co-administration of low-
dose (3 mg/kg) caffeine, a non-selective adenosine receptor antagonist, augments 
respiratory and grip function to a greater degree than AIH alone. Caffeine is rapidly 
absorbed, and then degraded to paraxanthine in the liver via Cytochrome P450 1A2 
(CYP1A2). Maximal serum concentrations are reached within ~1 hour of 
consumption.(42) The plasma half-life of caffeine is ~4 hours, but ranges from 2-8 hours 
between individuals.(43) Caffeine freely crosses the blood-brain barrier and enters the 
CNS; 3 mg/kg of caffeine inhibits A2A (~60%) and A1 receptors (50%); 6 mg/kg of caffeine 
yields ~75% A2A and ~65% A1 receptor inhibition.(44) Caffeine also inhibits 
phosphodiesterase activity, but only at doses >9 mg/kg.(45) Caffeine is the most widely 

used psychoactive drug worldwide (46), and is considered safe for use in most 
neurodegenerative disorders, including ALS (47, 48). 
Preliminary studies in rodent models of ALS: 
Diminished breathing ability can be partially restored by harnessing the intrinsic capacity 
for plasticity (1). In rats with incomplete cervical spinal injury, even a single presentation 
of AIH increases phrenic/diaphragm function ipsilateral to injury (22, 49). Repetitive AIH 
for one week enhances both the magnitude and duration of this effect (20, 31, 35). The 
mechanism of functional recovery is a shifting function of time post-injury; for example, 1 
week of daily AIH beginning 1 week post-injury improves breathing capacity by an 
adenosine dependent, serotonin-independent mechanism (31, 50). On the other hand, 1 
week of daily AIH elicits functional improvement of breathing capacity by a serotonin-
dependent, adenosine constrained mechanism. Thus, multiple, time-dependent 
mechanisms can contribute to the functional plasticity observed.  Both mechanisms of 
functional recovery are transient, although prolonged repetitive AIH exposures appear to 
elicit more robust and prolonged functional recovery. We are currently exploring optimal 
protocols with respect to both the amplitude and duration of functional improvement.  
Preliminary studies demonstrate that even a single AIH exposure restores 
diminished phrenic nerve activity during late-stage disease in a rat model of ALS, the 
SOD1G93A transgenic rat (10, 11, 51, 52). Indeed, in end-stage rats, AIH-induced phrenic 
motor facilitation is nearly double that found in wild-type littermates or in mutant rats 
during early stage disease (52). In end-stage ALS rats, enhanced AIH-induced phrenic 
motor facilitation utilizes the same serotonin-dependent mechanisms as in wild-type rats 
(10). These preliminary data and proof of concept studies in humans with incomplete 
spinal cord injury provide a strong rationale to evaluate responses to a single session of 
AIH in patients with ALS. Our initial investigations on a single AIH presentation will guide 
efforts as we work to evaluate repetitive AIH as a potential therapeutic modality in humans 
with ALS.  
 
Our plan:   
As a relentlessly progressive neuromuscular disease that leads to paralysis from motor 
neuron death, any strategy to preserve motor function for as long as possible will greatly 
impact the quality of life in those suffering from ALS. Current clinical ALS management 
includes various forms of assisted ventilation (53). However, assisted ventilation 
atrophies respiratory muscles, further degrading independent breathing ability (54); it 
takes things in the wrong direction. AIH is a potential therapy to preserve independent 
breathing ability, delaying the need for assisted ventilation and improving the quality and 
duration of life for ALS patients. We begin this line of investigation with two, limited specific 
aims designed to establish proof-of-principle that AIH has the intended effect. 
 We will begin investigations of AIH in ALS patients by focusing on a single AIH 
session to establish proof-of-principle before considering more extensive studies 
of repetitive AIH, which has greater potential to alter the course of disease. 
  Since the major cause of death in ALS is ventilatory failure (34), and studies of 
AIH-induced respiratory motor plasticity led to the principle repeated AIH as a 
therapeutic modality (9), our initial focus will on AIH and breathing ability in ALS 
patients.  

 We will collect additional preliminary data on limb function (grip strength) since AIH 
increases strength in several motor systems, potentially benefiting patients.  
 Aim 3 (optional aim) will independently evaluate the effect of pre-treatment with 
caffeine on respiratory and non-respiratory motor performance post-AIH. 
 Our goal with this initial project is to collect preliminary data necessary to provide 
proof-of-concept, and to provide the foundation for extramural grant applications.  
Specific Aims 
Aim 1: Test the hypothesis that a single AIH trial increases maximal respiratory and 
non-respiratory motor function in ALS.  Dependent variables will include: maximal 
inspiratory pressure (MIP) and maximal handgrip force (MHGF). We will address the 
following questions: 
 
a. Does AIH increase voluntary inspiratory pressure generation?  
b. Does AIH increase maximal handgrip force?   
c. Are baseline MIP and MHGF inversely related to AIH-induced facilitation?  
 
Aim 2: Test the hypothesis that a single AIH trial increases resting respiratory 
motor function in ALS. Dependent variables will include: resting tidal volume, 
respiratory EMG at rest, resting ventilatory drive. Three questions will be addressed:  
a. Does AIH increase resting tidal volume?  
b. Does AIH increase the resting ventilatory drive? 
c. Does AIH increase respiratory muscle activation and restore more normal 
respiratory muscle recruitment patterns? 
Aim 3: Test the hypothesis that pre-treatment with caffeine will augment respiratory 
and non-respiratory motor function. Dependent variables will include: maximal 
respiratory pressures, maximal grip force, resting tidal volume, EMG. Three questions will 
be addressed:  
a. Does acute consumption of caffeine enhance AIH-induced facilitation of 
respiratory and non-respiratory motor function?  
b. Is there a difference in AIH-induced facilitation of respiratory and non-
respiratory motor function between slow and fast caffeine metabolizers? 
c. Does acute consumption of caffeine improve the AIH-induced increase in          
respiratory muscle activation? 
 
Research Plan 
Design: Subjects will be 
screened for eligibility on 
Day 1 (Figure 1); those 
who meet the criteria will 
continue with the study.  
 
The study design (Figure 2) will consist of up to three separate testing sessions, 
separated by approximately two weeks (+/- 3 days). One session will be a single, ~45-
minute AIH protocol. The other will be 45 minutes of Sham AIH (ie. normoxia). Session 
order will be random. A third, optional session will be offered to eligible subjects (caffeine 
+ AIH). Each session is expected to last ~3 hours, including equipment setup, patient 
positioning and pressure relief, rest breaks, AIH/Sham AIH, and post-intervention tests. 
Figure 1: Proposed screening procedures.  
Overnight sleep monitor (optional)
Initiate study procedures
Not Eligible for Participation
Screen Failure:
Informed 
Consent
• O2 use
• FVC <60% pred.
DAY 1: ELIGIBILITY SCREEN
Screen Pass: 
• Medical screen 
• Forced vital 
capacity (FVC) 
test
• Meets all 
eligibility criteria 

Subjects who elect to participate in Aim 3 will be scheduled for a third study session 
consisting of administration of 3 mg/kg of caffeine, followed by AIH 1 hour after caffeine 
consumption. 
Study outcomes in Aim 1 include strength tests (respiratory and handgrip; MIP and 
MHGF), respiratory drive (P0.1), tidal breathing, and the hypoxic ventilatory response 
(HVR). In Aim 2, respiratory muscle recruitment will be tested at normoxic rest, during 
hypoxia, and with maximal voluntary inspiratory pressure generation. Aim 3 outcomes 
include maximal respiratory pressures, MHGF, tidal breathing, and saliva tests of caffeine 
metabolism. 
 
Subjects: We propose to study 
18 ALS patients and up to 30 
healthy age/sex matched control 
subjects. Patients who meet 
study criteria will be identified by 
Dr. Wymer (Co-I) through the UF 
adult neuromuscular clinic. The 
study PI will screen and consent 
patients. The PI will review the 
eligibility 
criteria, 
study 
procedures, participation risks, 
and the ability of subjects to 
withdraw from the study at any 
time.  
 
Eligibility Criteria. 
Subjects will be eligible to participate if they have a clinical diagnosis of ALS or are a 
healthy adult; are between 21 and 75 years of age; and have a baseline FVC >60% 
predicted for age, sex and height. Baseline FVC is routinely performed at every clinic for 
ALS patients. The AIH study team (PI or coordinator) will email a PFT report request to 
the clinic respiratory therapist, in advance of clinic. On the day of clinic, the respiratory 
therapist will upload the requested PFT reports directly into EPIC. After clinic, the AIH 
study coordinator will locate the PFT report in EPIC and save it to the electronic study 
binder. If the PFT report indicates the FVC is within study criteria, the subject can be 
contacted for an appointment. If the FVC falls below study eligibility, the patient will not 
be scheduled for a screening visit. 
Patients will be classified into two groups: 1) uses no form of external respiratory 
support (IND); and 2) and uses non-invasive external respiratory support while sleeping 
for at least 2 nights per week to assist night time breathing or treat sleep apnea (NIV). 
Since 4-6 subjects in each group may fail to meet all eligibility criteria or attend the second 
testing session, we request to enroll up to 18 patients with ALS, with the goal of 
completing all study procedures in 12 patients. In addition, we request enrollment of up 
to 30 healthy controls to complete all study procedures in 20 (up to 20 healthy controls 
may be needed as a reference set for the EMG data analysis described on page 10). The 
healthy controls will be recruited via listings for this study on CT.gov and the 
StudyConnect website. 
Fig 2 Proposed study design.  
 
 

 
Patient and control participants are ineligible if they: are pregnant, have diagnosed 
cardiovascular disease, have a BMI >35 kg/m2, currently take selective serotonin 
reuptake inhibitors (SSRI), have a history of seizures, have a history of hospitalization for 
sepsis, had a respiratory infection or took antibiotic medications within the past 4 weeks, 
use external respiratory support during any waking hours, participate in a pharmaceutical 
trial to treat ALS, or have any other medical condition the PI (Smith) or medical director 
(Wymer) identify would make it unsuitable to participate.  
Additionally, subjects will be ineligible to participate in Aim 3 (caffeine+AIH) if they: 
report negative side effects to caffeine consumption (e.g. anxiety, dizziness); have a 
diagnosis of diabetes (due to effect of caffeine on glucose metabolism (55) or hepatic 
cirrhosis (impaired metabolism of caffeine (56), or take antiarrhythmic or bronchodilator 
medications (57). 
 
The sample size is consistent with the time and budget required to acquire strong 
pilot data for an NIH R01 grant, which is the major goal of this project.  
 
Eligible subjects who agree to participate and sign the informed consent form 
approved by UF will undergo further screening procedures prior to beginning study 
procedures. Screening will include a detailed medical and pharmacological history, 
caffeine consumption questionnaire, forced vital capacity (FVC) testing and resting 3-lead 
ECG. The likelihood of obstructive sleep apnea will be evaluated using the STOP-BANG 
questionnaire. Subjects will be informed of their STOP-BANG questionnaire results, and 
if sleep apnea is suspected, then they will be referred for possible clinical treatment (which 
is outside of the scope of this study). If they choose to pursue clinical treatment for sleep 
apnea, then they will be included in the study after 3 months of treatment have elapsed. 
If they choose not to seek treatment, they will be included in the study. This plan is 
consistent with recommendations of a recent Intermittent Hypoxia workshop considering 
the inclusion/exclusion of subjects with sleep apnea; recommendations were made in 
based on input from two nationally known sleep apnea experts participating in that 
workshop (Drs. David Gozal, University of Chicago, and S. Badr at Wayne State 
University).  
At the screening session, subjects who meet all study criteria will be invited to 
participate in a third study session consisting of caffeine + AIH. Subjects who decline to 
participate in Aim 3 remain eligible to participate in Aims 1-2. 
 
Screening Tests  
 Forced vital capacity – the maximal volume of gas that can be forcefully exhaled, 
following full inspiration. FVC is a standard measure for clinical care and research, but 
lacks sensitivity in mild neuromuscular diseases, including ALS (58). Upright FVC will 
be conducted as per published American Thoracic Society and European Respiratory 
Society (ATS/ERS) guidelines (59-61). Study team will conduct FVC testing on 
screening/study visit #1 when any of the following conditions are present: (1) the most 
recent VC is <63% predicted, (2) most recent VC is >30 days from screening, or (3) 
written verification of the clinic VC is unavailable. Individuals with an FVC <60% of 
predicted will be ineligible for further participation in this study.  
 3-lead EKG – cardiac rate and rhythm will be evaluated. The presence of resting 
tachycardia (HR>110 bpm), atrial fibrillation or other dysrhythmias will exclude 

subjects from participation.  
 Sleep apnea screening. Subjects  
o STOP-BANG questionnaire - a validated screening tool for OSA (62). All subjects 
must complete the STOP-BANG questionnaire, which consists of 4 yes-no 
questions regarding the presence of symptoms of OSA, plus 4 questions regarding 
known physical risk factors for obstructive sleep apnea. Individuals who present 
with a high risk for OSA and are currently not receiving treatment (e.g. overnight 
positive pressure ventilation or mouthpiece) will be referred to a clinical sleep 
specialist for further evaluation.  
o Overnight sleep monitor – subjects will have the option to wear an overnight sensor 
that non-invasively monitors arterial oximetry and actigraphy (WatchPAT, Itamar 
Medical), to estimate the severity of sleep-disordered breathing. Home-based 
peripheral arterial tonometry has a high specificity/sensitivity for detecting OSA 
(63). If clinically-significant OSA is detected by the sleep monitor (an apnea-
hypopnea index >10 events/hour), subjects will be referred to a clinical sleep 
specialist for further evaluation.  
o Caffeine consumption questionnaire- subjects will be asked about their usual daily 
caffeine consumption habits. We will also ask the subjects about their possible 
reactions after consuming caffeine. This questionnaire will help us understand the 
responsiveness of the subjects to caffeine and help rule out individuals with 
adverse reactions to caffeine. 
 
Independent Variable - Acute Intermittent Hypoxia (AIH) vs Sham AIH 
Subjects will undergo a single session of AIH and a single session of Sham AIH (Figure 
3). Test sessions will be scheduled two weeks apart at the same time of day. Subjects 
will be instructed to avoid caffeine and nicotine products for >6 hours prior to and avoid 
eating within 30 minutes of test 
sessions. Subjects will sit upright in a 
chair with the head and trunk 
supported while breathing through a 
sealed face mask. The mask will be 
connected to a pneumotachograph 
and 
pressure 
transducer 
(Hans 
Rudolph) to measure respiration, and 
a commercially-available hypoxicator 
(Hypoxico, Inc.) will provide either 
hypoxic or normoxic gas mixtures. 
Respiratory rate, end-tidal CO2, heart 
rate, SpO2, and respiratory muscle 
EMG will be continuously monitored during the testing sessions (PowerLab, 
ADInstruments).  
 
1. AIH.  A single, 45-minute AIH session will consist of 15, 1 minute hypoxic 
episodes (O2=9-12%) separated by up to 2 minutes of normoxia (O2 =21%) (to 
enable SpO2 to return to at least 92% for 1 full minute). The oxygen content of 
the hypoxic gas will be set at 9%, and adjusted upwards if necessary to maintain 
Fig 3. Schematic of AIH and Sham AIH interventions. 
Each AIH session will consist of 1-minute intervals of 
hypoxia (9-12% O2, shown in blue) separated by up to 2-
minute intervals of normoxia. Sham AIH will use identical 
equipment, but the hypoxicator machine will deliver room 
air continuously. 

SpO2 above 78% for safety reasons. We do have reasons to believe that the 
specific SpO2 within this range is an inverse indicator of therapeutic efficacy 
since the proximate stimulus to plasticity is activation of carotid body 
chemoreceptors (with neural activation) versus tissue hypoxia per se. If SpO2 
levels below 78% are observed, the hypoxic episode will end early and the 
hypoxicator will be adjusted to raise that value prior to the next episode. Healthy 
individuals and patients with spinal cord injury typically have difficulty in 
distinguishing room air from the mild hypoxic conditions proposed here.  
2. Sham AIH.  Sham AIH will consist of 45 minutes of normoxic gas delivered by the 
hypoxicator through the identical facemask system with identical procedures 
(other than the level of inspired oxygen per se).  
3. Caffeine + AIH. Following baseline testing, subjects will consume 3 mg/kg of 
caffeine. Caffeine capsules will be prepared for each subject based upon the 
weight recorded at the screening visit. AIH will then commence in the same pattern 
as described above beginning 1 hour after caffeine consumption. 
 
 
 
Aim 1 Dependent Variables – Maximal Voluntary Contractions   
 Maximal respiratory pressures – maximal voluntary static contractions of the 
inspiratory or expiratory muscles against a closed valve, measured at the mouth (64). 
Sniff nasal inspiratory pressure measures inspiratory force generation at the nose. 
This more natural maneuver has lower variability and learning effects (65) and 
appears to decline in conjunction with the need for nocturnal ventilation in ALS (66, 
67). The test will be repeated until 3 measurements are obtained within 10% 
variability; a minimum 20-second rest will be provided between measurements. 
Maximal inspiratory pressures will be measured at baseline and 60 min post-AIH.  
 Peak cough flow -  the maximal expiratory airflow measured through a facemask 
during a maximal-effort voluntary cough. Subjects will repeat 3-5 voluntary coughs to 
achieve 3 measurements within 10% variability. The peak cough flow will be measured 
at baseline and 30 min post- intervention. 
 Maximal voluntary grip force – maximal static voluntary handgrip contractions will be 
evaluated in a seated position with the arm at the side and elbow flexed to 90 degrees. 
The test will be repeated until 3 measurements are obtained with <10% variability, and 
a minimum 15-second rest between measurements. Significant gains in isometric 
force have been reported with a single AIH session in patients with spinal cord injury. 
Grip strength will be measured at baseline, 30 and 60 minutes post-intervention. 
 
Aim 2 Dependent Variables – Resting/automatic respiratory motor function  
 Minute ventilation – the pneumotachograph and pressure transducer connected to the 
face mask will record breath-by-breath flow, volume, mouth pressure, and breathing 
rate. After achieving a stable tidal volume, 5 minutes of tidal breathing will be recorded. 
Tidal breathing will be measured at baseline, during hypoxic episodes, immediately 
following AIH, and at 30 and 60 minutes post-intervention. 
 Ventilatory drive. Respiratory drive will be estimated with pressure generation against 
a transiently occluded airway in the first 0.1 sec of inspiration (ie. P0.1); subjects will 

be seated upright and breathing quietly through the face mask. An inspiratory valve 
will be manually occluded during the expiratory phase of a breath and will be 
maintained until the end of the subsequent inspiratory effort. Five measurements will 
be conducted, with 5-15 un-occluded breaths between each P0.1 measurement. This 
validated test is resistant to learning or sensory bias and reflects unaltered 
neuromuscular effort (68, 69). P0.1 will be measured at baseline, 30 and 60 minutes 
post-intervention. 
 Respiratory muscle EMG. Surface EMGs of the respiratory muscles (up to six 
muscles, bilaterally: scalene, sternocleidomastoid, 2nd parasternal, 5th external 
intercostal, 8th external intercostal, and diaphragm) will be recorded during the test 
session. The root mean square (RMS) of each muscle will be averaged. Multi-muscle 
activation will be calculated using a validated, vector-based analysis(70) that provides 
a magnitude (Response Vector) and pattern (Similarity Index) of composite EMG 
activity. We will then compare multi-muscle activity from each ALS subject to the 
Prototype Response Vector calculated as the average Response Vector and Similarity 
Index calculated from the pooled control subjects (71). 
 
Aim 3 Dependent Variables 
 Maximal respiratory pressures – as described in Aim 1 
 Maximal voluntary handgrip force – as described in Aim 1 
 Tidal breathing pattern – as described in Aim 2  
 Markers of caffeine metabolism and adenosine-mediated AIH. Saliva specimens 
will be collected at the screening visit and processed for single nucleotide 
polymorphisms in three specific genes. Polymorphisms in CYP1A2 (rs762551), 
ADORA2A (rs5751876), and BDNF (rs6265) will be tested using a commercially 
available TaqMan assay with PCR instrument and genotyping analysis software. 
 Salivary caffeine – Saliva specimens will be collected before baseline testing and 
60 minutes post-AIH, to measure changes in salivary caffeine levels. Salivary 
caffeine will be analyzed using a HPLC assay. The caffeine powder used by 
Westlab Pharmacy will be available to UF’s ICBR to create a caffeine standard for 
assay analysis. 
 
Statistical Analysis  
The outcomes for both aims are largely quantitative and continuous. Sample distributions 
and summary statistics will be calculated for each outcome, and non-linear data 
distributions will be transformed as needed. Study outcomes for Aims 1-2 include acute 
changes in voluntary strength, resting ventilation, and respiratory drive. Each of these will 
be evaluated with 3-way repeated measures ANOVAs. Factors will be subject group 
(patient vs control), AIH condition (AIH vs Sham AIH), and time (pre, 0 minutes post, 30 
minutes post, 60 minutes post); Tukey’s post hoc test will be used to assess individual 
differences. A separate ANOVA will compare the independent effect of caffeine on motor 
function 60 minutes post-AIH. We will work with BERD to use genetic testing results to 
model independent effects of caffeine metabolism genotype on AIH motor facilitation, as 
pilot data to estimate sample size for a larger, future project. For the analysis of sEMG 
Response Vector and Similarity Index, baseline FVC will be used as a covariate. These 
will be tested with 2-way repeated measures ANOVAs (AIH only, factors: subject group 

and time). Relationships between variables will be evaluated with Pearson’s correlation. 
Statistical significance will be p<0.05. 
 
Possible Discomforts and Risks 
The physiological (and perceptual) effects of intermittent hypoxia in humans are well 
studied at the degree and duration of hypoxia as proposed here, and appear safe and 
well-tolerated (7, 33). Similar AIH protocols have been shown to improve ventilation (72, 
73), enhance cerebral blood flow (26), and increase muscle sympathetic nerve activity. 
More severe hypoxia (3-6% O2) and recurrent (>>15 episodes per day) have been 
associated over time with hypertension, increased systemic inflammation and 
neurocognitive deficits, as seen clinically in obstructive sleep apnea (8, 74, 75). On the 
other hand, intermittent hypoxia protocols consisting of less severe (>9% O2) and 
recurrent (<15 episodes/day) are associated with the opposite outcome, including 
reduced blood pressure, anti-inflammatory effects and neurocognitive enhancement (8, 
74, 75). The hypoxia dose and duration have been carefully selected to optimize impact 
while minimizing known risks. We will monitor subjects continuously to detect adverse 
events, if any. 
 
While this AIH protocol is well-tolerated and appears safe in most individuals, some 
patients may report transient light-headedness, dizziness, or reduced vision. Some 
patients also report an increase in respiratory sensation (mild to moderate shortness of 
breath), or rarely notice an increase in breathing (tidal volume) during the hypoxic periods. 
During and after the AIH protocol, we continuously monitor heart rate, respiratory rate, 
end tidal CO2 (ETCO2) and oxygen saturation (SpO2) as well as the rating of dyspnea. 
The FiO2 will be closely titrated to maintain SpO2 >78% during hypoxic episodes. While 
excessive HR and BP responses have not been reported, AIH will be stopped in the 
unlikely event the heart rate increases or decreases >30% from the resting value, and/or 
systolic blood pressure changes more than 30% from the resting value. Subjects will be 
informed that they can request to stop the study at any time. If SpO2 falls below 75%, the 
AIH intervention will be terminated and supplemental O2 provided. 
 
Additionally, the mask interface for administering AIH and measuring breathing can feel 
warm, moist, and/or confining to some individuals. To offset discomfort, the mask will be 
removed during rest periods and between post-AIH measurements. We prefer to use a 
mask interface, since subjects are able to easily communicate while using a mask and it 
can accommodate patients with bulbar/oral weakness. However, if subjects cannot 
tolerate the mask, a mouthpiece and nose clip can be offered as an alternative.  
 
Neurologic effects of low- to moderate-dose caffeine (3-6 mg/kg) include increases in 
alertness, mood, reaction time, and motor function (limb and possibly respiratory muscle 
strength, gait). (76-79). Higher doses of caffeine elicit concurrent A1 receptor competitive 
inhibition, which may increase heart rate and/or blood pressure. However, both population 
studies and controlled studies in heart failure indicate caffeine has either a neutral or 
protective effect on cardiovascular risk (80, 81) as well as arrhythmias (82), in acute doses 
up to 500 mg (83). Caffeine may promote feelings of anxiety or fear in some individuals. 
Caffeine-induced anxiety has been linked to known polymorphisms of the ADORA2A 
gene and is associated to an aversion of chronic caffeine consumption (84). Caffeine is 
also a weak, non-specific phosphodiesterase inhibitor. While phosphodiesterase 

inhibition occurs only at high doses (>9 mg/kg) that exceed typical human 
consumption(45, 85), some individuals report stomach pain or queasiness after 
consuming caffeine.  
 
To minimize possible side effects of caffeine, we will exclude subjects with known 
sensitivity to caffeine. This study will use a standard dose of 3 mg/kg, which is considered 
by FDA and EFSA (European Food Safety Authority)  (86) to be a safe and well-tolerated 
quantity in most individuals. Additionally, caffeine is generally recognized as safe (GRAS) 
by FDA's regulations solely in cola-type beverages up to 0.02% (200 ppm). 21 C.F.R. § 
182.1180. 
The caffeine capsules will be compounded by Westlab Pharmacy, a PCAB Accredited® 
compounding pharmacy located approximately 2 miles from the University of Florida’s 
campus. Capsules will contain caffeine powder and filler (cellulose gum) in order to help 
in the breakdown and absorption of the caffeine. Westlab Pharmacy will compound 
individual capsules at a dose corresponding to 3 mg caffeine per kg body weight. To meet 
the minimum compounding requirements of the external pharmacy, we will order 3-5 
capsules corresponding to several pre-determined body weights (e.g. 5, 10, 25, 50, 60, 
70, 80, 90, and 100 kg). Westlab Pharmacy will then provide subjects with the minimum 
number of capsules needed to approximate (but not exceed) a 3 mg/kg dose. Each 
subject will orally ingest the caffeine capsules with water.  
 
Respiratory strength testing procedures increase voluntary respiratory drive of subjects, 
which in some patients can result in a briefly elevated heart and/or respiratory rate, and 
decreased SpO2, feelings of shortness of breath and exertion. Some individuals find the 
transient exertion caused by breathing tests to be uncomfortable or anxiety-provoking 
(87). These effects occur infrequently and resolve upon removal of the respiratory 
stimulus. Nevertheless, we will monitor vital signs during the respiratory tests and be 
certain that patients understand they can rest as long as they like between test bouts or 
stop any test at any time. 
 
The surface EMG electrodes are secured to subjects with a double-sided adhesive tape, 
which can cause skin redness or irritation in some individuals with adhesive sensitivities. 
Subjects will be asked about any known skin sensitivities, and if present a hypo-allergenic 
tape will be used.  
 
The sensors attached during the ECG and overnight sleep monitoring may also be 
uncomfortable and cause mild skin irritation in some individuals sensitive to adhesive 
tape. The study team will attach the ECG stickers. A hypo-allergenic tape will be used 
instead for these individuals. 
 
Patients with ALS will be recruited from the UF ALS clinic, which encompasses a large 
catchment area. The study risks of travel for patients with neuromuscular disease include 
fatigue, muscle and joint pain, and difficulty with pressure relief and toileting regimens. In 
addition, test performance may be diminished by fatigue. Up to $200 reimbursement has 
been budgeted for the cost of overnight lodging for patients.  
 
For testing of caffeine metabolism genes, risks are extremely small but include the risk of 
exposing a subject’s genetic information. To offset these risks, we will de-identify the 

samples, and focus upon specific single nucleotide polymorphisms (SNP) in 3 genes that 
have essential known functions relating to caffeine metabolism (CYP1A2 and ADORA2A) 
and synaptic plasticity (BDNF). Caffeine metabolism genes and salivary caffeine levels 
will be analyzed at UF’s ICBR, to minimize exposure of a subject’s genetic information. 
 
Safety oversight will occur internally through the UF IRB-01 as well as through a data 
safety monitoring board (DSMB). The DSMB will be convened before enrollment of the 
first patient, after completion of the first three patients, and at least annually thereafter. 
The DSMB will include three members - 1 expert in intermittent hypoxia, one in 
sleep/pulmonary medicine, and one in rehabilitation. At least 1 member will be external, 
since a wealth of external expertise in intermittent hypoxia expertise is available and this 
issue is of great interest to the scientific community. Notes will be taken and available for 
the IRB at continuing review.  
Testing and enrollment of subjects will be paused and the DSMB will be convened, if any 
of the following events occur: (1) a clinically significant respiratory event determined by 
the PI to be related to study procedures, (2) a clinically significant cardiovascular event 
determined by the PI to be related to study procedures, (3) a serious adverse event as 
defined by IRB-01. Any subsequent study activities would only be resumed when cleared 
or modified by the IRB-01 and DSMB. 
 
Possible Benefits 
The expected outcome of this study is an increased knowledge of the effects of AIH on 
short-term maximal muscle recruitment/contraction and minute ventilation. No direct 
benefit is anticipated for the participants. Based upon this increased knowledge of 
respiration and AIH, we hope to evaluate the feasibility for studying longer-term benefits 
of AIH on breathing in patients in ALS. This could eventually lead to the investigation of 
future treatments for respiratory problems in motor neuron diseases such as ALS. 
 
Conflict of Interest 
None of the investigators have identified a conflict of interest. 
 
 

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