J Appl Physiol 122: 1398–1405, 2017.

First published February 23, 2017; doi:10.1152/japplphysiol.00885.2016.

RESEARCH ARTICLE

An international collaboration studying the physiological and anatomical

cerebral effects of carbon dioxide during head-down tilt bed rest: the
SPACECOT study
X Karina Marshall-Goebel,1,2,4 Edwin Mulder,1 Dorit Donoviel,3 Gary Strangman,3,4 Jose I. Suarez,3
Chethan Venkatasubba Rao,3 Petra Frings-Meuthen,1 Ulrich Limper,1 Jörn Rittweger,1,5
Eric M. Bershad,3 and the SPACECOT Investigators Group
1
Institute of Aerospace Medicine, German Aerospace Center, Cologne, Germany; 2Faculty of Medicine, University of
Cologne, Cologne, Germany; 3Baylor College of Medicine, Houston, Texas; 4Neural Systems Group, Massachusetts General
Hospital, Harvard Medical School Charlestown, Massachusetts; and 5Department of Pediatrics and Adolescent Medicine,
University of Cologne, Germany
Submitted 3 October 2016; accepted in final form 17 February 2017

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Marshall-Goebel K, Mulder E, Donoviel D, Strangman G,
Suarez JI, Venkatasubba Rao C, Frings-Meuthen P, Limper U,
Rittweger J, Bershad EM, the SPACECOT Investigators Group.
An international collaboration studying the physiological and an-
atomical cerebral effects of carbon dioxide during head-down tilt
bed rest: the SPACECOT study. J Appl Physiol 122: 1398 –1405,
2017. First published February 23, 2017; doi:10.1152/japplphysiol.
00885.2016.—Exposure to the microgravity environment results in
various adaptive and maladaptive physiological changes in the human
body, with notable ophthalmic abnormalities developing during 6-mo
missions on the International Space Station (ISS). These findings have
led to the hypothesis that the loss of gravity induces a cephalad fluid
shift, decreased cerebral venous outflow, and increased intracranial
pressure, which may be further exacerbated by increased ambient
carbon dioxide (CO2) levels on the ISS. Here we describe the
SPACECOT study (studying the physiological and anatomical cere-
bral effects of CO2 during head-down tilt), a randomized, double-
blind crossover design study with two conditions: 29 h of 12°
head-down tilt (HDT) with ambient air and 29 h of 12° HDT with
0.5% CO2. The internationally collaborative SPACECOT study uti-
lized an innovative approach to study the effects of headward fluid
shifting induced by 12° HDT and increased ambient CO2 as well as
their interaction with a focus on cerebral and ocular anatomy and
physiology. Here we provide an in-depth overview of this new
approach including the subjects, study design, and implementation, as
well as the standardization plan for nutritional intake, environmental
parameters, and bed rest procedures.
NEW & NOTEWORTHY A new approach for investigating the
combined effects of cephalad fluid shifting and increased ambient
carbon dioxide (CO2) is presented. This may be useful for studying
the neuroophthalmic and cerebral effects of spaceflight where ceph-
alad fluid shifts occur in an elevated CO2 environment.
microgravity; visual impairment; analog; intracranial pressure; space-
flight
EXPOSURE TO MICROGRAVITY RESULTS in multiple physiological
and anatomical changes, some of which may alter cerebral and
ocular structure and function. Currently, more than one-half of
Address for reprint requests and other correspondence: E. Bershad, Baylor
College of Medicine, One Baylor Plaza, Houston, TX 77030 (e-mail: bershad
@bcm.edu).
1398 8750-7587/17 Copyright © 2017 the American Physiological Society
astronauts present with ophthalmological changes during 6-mo
missions on the International Space Station (ISS). The National
Aeronautics and Space Administration (NASA) has identified
this as a high-priority risk to human health in space and named
it spaceflight-induced intracranial hypertension/vision altera-
tions, also referred to as the visual impairment and intracranial
pressure (VIIP) syndrome (1) or microgravity ocular syndrome
(8). The findings include swelling of the optic nerve head,
increased optic nerve sheath diameter, choroidal folds, cotton
wool spots, flattening of the posterior eye globe, and hyperopic
shifts up to 3 diopters (5, 7). Although the etiology of these
ocular changes is currently unknown, there are similarities to
patients with idiopathic intracranial hypertension. Several as-
tronauts with papilledema also had mildly elevated intracranial
pressure (ICP) measured postflight through lumbar puncture
(7). These findings have prompted the hypothesis that ICP is
increased during spaceflight secondary to a microgravity-in-
duced headward fluid shift and decreased cerebral venous
outflow due to the loss of physiological hydrostatic pressure
gradients in space.
Because the ISS is a closed environment, ambient carbon
dioxide (CO2) levels average 0.5%, i.e., approximately 10
times higher than terrestrial levels (0.04%) (6). However, with
no natural convection currents in microgravity, large fluctua-
tions in atmospheric CO2 occur over hours and days, and
pockets of high CO2 concentrations may form in areas of low
ventilation (14). As a potent arterial vasodilator, CO2 can
evoke manifold physiological responses. Notably, hypercapnia
increases cerebral blood flow (9) and may lead to acutely
increased intracranial volume and thus elevated ICP. There-
fore, exposure to increased ambient CO2 may exacerbate the
hypothesized microgravity-induced increase in ICP during
spaceflight (6). For decades, head-down tilt (HDT) bed rest has
been used as a ground-based microgravity analog for physio-
logical research (10a, 20). However, HDT bed rest studies
performed in the 6° HDT position with ambient air do not
reproduce most of the cerebral or ophthalmic findings seen in
astronauts (17, 18). Conversely, short-duration exposure to
steeper HDT angles have demonstrated decreased cerebral
venous outflow (9) and increased ICP (4, 11), consistent with
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 The SPACECOT Study • Marshall-Goebel K et al.
hypothesized contributing factors to microgravity-induced oc-
ular changes. Here, we present a novel ground-based approach
to study two of the major hypothesized contributing factors to
ocular changes in space: headward fluid shifting and elevated
ambient CO2, as well as their possible interaction. Because this
study was the first to implement this new approach to HDT bed
rest, vital signs, blood parameters, and standardization tech-
niques are presented as standard measures.
Overall, our aim was to perform a comprehensive evaluation
of the cerebral, ocular, and cardiopulmonary physiological
effects of HDT with and without elevated ambient CO2. As a
secondary aim, we evaluated the effects of short-duration (3 h)
exposure to 3% CO2 as a simulation of CO2 accumulation that
may occur on the ISS. The SPACECOT Study (studying the
physiological and anatomical cerebral effects of CO2 during
head-down tilt) was an international collaboration of more than
10 institutions and, to our knowledge, the first study to inves-
tigate the combined effects of HDT and increased ambient CO2
for ⬎24 h. This approach was accompanied by a suite of both
gold-standard and innovative noninvasive monitoring technol-
ogies to provide a comprehensive understanding of the HDT ⫹
CO2 challenge on whole body physiology. In this overview
paper, we focus on the framework of our new approach and
present baseline subject characteristics and general results.
Specific results related to the various procedures and physio-
logical systems will be presented in subsequent papers.
MATERIALS AND METHODS
The SPACECOT study was performed at the : envihab facility at
the German Aerospace Center in Cologne, Germany. The :envihab is
a state-of-the-art environmental medicine research facility that allows
for full environmental and atmospheric conditioning of the bed rest
facility, which consists of 12 bedrooms, a common area, bed rest
compatible showers, bathrooms, a metabolic kitchen, laboratories, and
a control room. The study procedure was approved by the local ethical
commission of the regional medical association (Ärztekammer Nor-
drhein) as well as the Baylor College of Medicine Institutional
Review Board and was registered at ClinicalTrials.gov (identifier
number: NCT02493985). A test run of the study schedule and proce-
dures was conducted in May 2015, and the full study was performed
in June to July of 2015. All of the subjects provided written informed
consent at the German Aerospace Center in Cologne, Germany.
Subjects. The SPACECOT study included six healthy male subjects
(means ⫾ SD: age, 41 ⫾ 5 yr; weight, 82 ⫾ 7 kg; height, 177 ⫾ 4
cm). Baseline subject demographics and anthropometrics are shown in
Table 1. Body surface area (BSA) was calculated from the classical
equation of Du Bois and Du Bois (3) as follows: BSA ⫽ 0.007184 ⫻
W0.425 ⫻ H0.725, where W ⫽ weight of subject in kilograms and
H ⫽ height of subject in centimeters. Subject inclusion criteria con-
sisted of healthy male subjects, ages 30 –55 yr, body mass index of
20 –26 kg/m2, nonsmoking for at least 6 mo, no current prescription
Table 1. Baseline characteristics of subjects included in the SPACECOT study
Subject Age, yr Weight, kg Height, cm BMI, kg/m2 BSA, m2
1 47 82.5 174 27.2 1.97
2 43 91.4 183 27.4 2.13
3 39 68.8 175 22.6 1.83
4 33 80.1 178 25.4 1.98
5 39 85.0 180 26.2 2.05
6 43 84.5 173 28.2 1.99
Means ⫾ SD 41 ⫾5 82 ⫾7 177 ⫾ 4 26.2 ⫾ 2 1.99 ⫾ 0.1 17,330 ⫾ 3,724
BMI, body mass index; BSA, body surface area; Vol, volume.
J Appl Physiol • doi:10.1152/japplphysiol.00885.2016 • www.jappl.org
1399
medication use, and with an exercise capacity as well as cardiovas-
cular and mental health that approximately matches the astronaut
corps. Prior to inclusion in the study, all subjects underwent a
comprehensive medical screening. Subject exclusion criteria were as
follows: V̇O2max outside of the range of 30 – 60 ml·kg⫺1·min⫺1,
history of migraines, history of ophthalmological conditions (includ-
ing glaucoma, retinopathy, and cataracts), history of chronic back
pain, increased risk of thrombosis, history of kidney stones or kidney
disorders, and any neurological, cardiovascular, or psychiatric condi-
tions. Subjects also underwent psychological evaluations, including
the big five inventory and the Freiburger Persönlichkeitsinventar. The
recruitment process also included 1 h in the 12° HDT position,
including 10 min in a magnetic resonance imaging (MRI) scanner
while in the HDT position with a face mask, to test eligibility with
these conditions, as well as ultrasound screening of ophthalmic
arteries to ensure suitability for noninvasive ICP monitoring (Vit-
tamed, Kaunas, Lithuania).
Study design. The SPACECOT study implemented a randomized,
double-blind crossover design with two conditions: 26.5 h of 12°
HDT with ambient atmosphere and 26.5 h of 12° HDT with 0.5% CO2
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atmosphere (Fig. 1). At the end of each condition (while still in the
12° HDT position), subjects were also exposed to a 3% CO2 atmo-
sphere for 2.5 h through a mask and tank system, to determine the
effects of short-term exposure to high CO2 concentrations, similar to
what astronauts may experience in areas with poor ventilation on the
ISS, leading to a full 29 h of HDT bed rest. The two HDT conditions
were preceded by a baseline data collection day that exhibited iden-
tical study procedures. The only exception was that, before the first
baseline condition, subjects came in 1 day prior for a familiarization
session with the facility, staff, techniques, and equipment involved in
the study. All baseline data were collected in the seated, upright
position. Exceptions include baseline flow-mediated dilation ultra-
sound and MRI-derived measures, which were collected in the supine
position, and several other parameters took baseline measures in both
the upright and supine positions. Between each HDT condition, there
was a washout period of 1 week to avoid carryover effects. The six
subjects were tested in two groups of three, with each group experi-
encing a different atmospheric conditioning order. Notably, both the
investigators and the subjects were blinded to the atmospheric condi-
tions during the two conditions and were unmasked after data analyses
were completed. In addition, : envihab technicians and engineers who
controlled and monitored atmospheric conditions offsite were not
scientifically involved in the study and operated independently from
study investigators.
All study procedures were in accordance with the standardization
plan issued by the European Space Agency and/or the International
Academy of Astronautics Study Group (15, 16). During the full 29 h
of bed rest, all subjects strictly adhered to the 12° HDT position
including during all daily activities such as eating, showering, and
going to the bathroom. Subjects were required to keep a daily log of
critical incidents and were not allowed a pillow of any kind during the
HDT phases of the study, as this would cause deviations from the full
body HDT position. Further, subjects were allowed to lie on their side
for short durations; however, one shoulder remained in contact with
Thorax Vol, cm3 Abdomen Vol, cm3 Thigh Vol, cm3 Calf Vol, cm3 Total Vol, cm3
18,911 9,937 2,707 1,579 27,483
21,846 12,999 7,422 2,259 41,208
12,863 5,856 3,648 1,423 23,006
13,331 6,555 5,021 1,771 26,915
16,550 7,698 3,793 1,926 27,987
20,477 10,493 3,778 1,850 31,733
8,923 ⫾ 2,706 4,395 ⫾ 1,656 1,801 ⫾ 290 29,722 ⫾ 6,276
1400 The SPACECOT Study • Marshall-Goebel K et al.
Fig. 1. The SPACECOT study was a randomized, double-blind crossover design study with two conditions: 29 h of 12° head-down tilt (HDT) with ambient air
and 29 h of 12° HDT with 0.5% CO2. Before each condition, subjects underwent 1 day of baseline data collection (BDC) in the seated, upright position.
the mattress at all times (sitting or standing not permitted), and the 12°
HDT angle was maintained. Each subject was accommodated in a
single-person room, and the daily schedule consisted of 6:30 AM
wake up and 10:30 PM lights out. Light intensity (100 lux) and
spectrum, enriched in green (~540 nm) wavelengths to entrain the
circadian system, were standardized and periodically verified with an
external spectral irradiance meter in the subject bedrooms and mea-
surement rooms. To track their activity during the nighttime as an
indicator of sleep quality, the subjects wore an Actiwatch activity
monitor (Actigraph, Pensacola, FL) on the wrist of the nondominant
arm.
Nutrition. All subjects received a strictly controlled diet, for the
entire duration of the study, tailored to individual resting metabolic
rates (RMRs; Table 2). The RMR was estimated based on body
weight and age from equations provided in the Human Energy
Requirements Report by the United States Food and Agriculture
Organization/World Health Organization/United Nations University
Expert Consultation (21). Then, for each individual, the daily energy
intake during stationary phases was set to 1.5 ⫻ RMR (corresponding
to light intensity daily physical activities), whereas during HDT the
daily energy content was set to 1.3 ⫻ RMR (corresponding to the
energy need of a sedentary adult). The carbohydrate content of the diet
was set to 50 –55% and dietary fat intake was set to 30 –35%, in line
with international bed rest standards (15). The selected food items
corresponded to a standard German diet. Fluid intake was regulated
and set to 42 ml·kg body wt⫺1·day⫺1 and, apart from the fluid portion
contained in the meals, was provided in the form of water and diluted
apple juice. Intake of caffeine, alcohol, and chocolate was prohibited.
All meals were planned and supervised by registered dieticians and
prepared in the : envihab metabolic kitchen. Nutrient content was
calculated using PRODI software (Kluthe Prodi 6.3 expert; Nutri-
science, Germany).
Measurements. As part of the standardized bed rest protocol,
general health indicators including three blood pressure and heart rate
readings (Intellivue MMS X2; Philips, Best, The Netherlands) were
obtained every morning in the fasted state immediately following the
scheduled wake-up at 6:30 AM. In addition, body mass was also
assessed daily in bed with a bed scale (DVM 5703; Sartorius,
Goettingen, Germany) following the first urine void of the day.
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Subjects were weighed in standardized dedicated clothing, the weight
of which was subtracted from the total weight for accurate subject
weight. Body temperature was also measured in the ear three times,
with the mean used for statistical analysis. Urine output was collected
and pooled every 24 h for assessment. Furthermore, venous blood
samples were taken at baseline (morning of HDT day, before entry
into 12° HDT position) and after 4.5 h HDT, 24 h HDT, and 28.5 h
HDT ⫹ 3% CO2. Various parameters were measured including white
blood cells (WBC), lymphocytes (LYM), monocytes (MON), neutro-
phils (NEU), eosinophils (EOS), basophils (BAS), red blood cells
(RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular
volume (MCV), mean corpuscular hemoglobin (MCH), mean corpus-
cular hemoglobin concentration (MCHC), red blood cell distribution
width (RDW), platelet count (PLT), and mean platelet volume (MPV).
The aforementioned measured parameters are presented as standard
health indicators, along with core environmental data.
To fully understand the physiological effects of HDT with and
without increased ambient CO2, a comprehensive set of cerebral,
ocular, and cardiopulmonary measurements were performed on a
carefully timed and adhered-to schedule. This ensured that measure-
ments with particular devices or assays were taken at the same time of
day across conditions for each subject as well as within 3 h of the
other subjects within the campaign group. Deviations from the
planned schedule were minimal (no more than 15 min), allowing for
cross-comparison and multimodality investigation. All data acquisi-
tion systems were time-synchronized to a master clock which was
displayed prominently, and the resulting individual data sets were
merged into a master data table in Stata v14. Results from the various
physiological and anatomical measurements will be published in a
series of discrete papers.
Statistical analysis. Statistical analysis was carried out in IBM
SPSS Statistics Version 20 (IBM, Armonk, NY). ANOVA and linear
mixed effect models were constructed with time and atmosphere as
main effects allowing for a time-atmosphere interaction, and subject
as a random effect. When a main effect was found to be significant,
Bonferroni post hoc analyses were implemented (adjusted for multiple
comparisons by dividing ␣ by the number of comparisons) to deter-
mine differences between time points and/or atmospheric conditions.
 The SPACECOT Study • Marshall-Goebel K et al. 1401
Table 2. Standardized nutritional intake for the three experimental days, including baseline data collection day, the first day
of 12° HDT, and the second day of 12° HDT
BDC 12° HDT (1st Day) 12° HDT (2nd Day)

Energy, kcal/day 2,649 ⫾ 133 2,287 ⫾ 114 2,656 ⫾ 143

Protein, g/day 98 ⫾ 9 97 ⫾ 8 97 ⫾ 9
Protein, g·kg body wt⫺1·day⫺1 1.21 ⫾ 0.00 1.20 ⫾ 0.01 1.20 ⫾ 0.01
Protein, %TEE/day 15 ⫾ 1 17 ⫾ 1 15 ⫾ 1
Fat, g/day 95 ⫾ 5 83 ⫾ 4 95 ⫾ 5
Fat, %TEE/day 33 ⫾ 0 34 ⫾ 0 33 ⫾ 0
Carbohydrates, g/day 333 ⫾ 13 274 ⫾ 12 336 ⫾ 15
Carbohydrates, %TEE/day 52 ⫾ 1 49 ⫾ 0 52 ⫾ 1
Fiber, g/day 33 ⫾ 1 31 ⫾ 1 33 ⫾ 2
Fluid, ml/day 3,393 ⫾ 305 3,407 ⫾ 304 3,405 ⫾ 301
Fluid, ml·kg body wt⫺1·day⫺1 42.0 ⫾ 0.0 42.0 ⫾ 0.0 42.0 ⫾ 0.0
Calcium, mg/day 1,068 ⫾ 26 1,152 ⫾ 43 1,133 ⫾ 63
Chloride, mg/day 6,311 ⫾ 472 6,478 ⫾ 506 6,598 ⫾ 587
Chloride, mmol·kg body wt⫺1·day⫺1 2.20 ⫾ 0.04 2.25 ⫾ 0.06 2.29 ⫾ 0.00
Potassium, mg/day 4,434 ⫾ 194 3,972 ⫾ 206 4,153 ⫾ 198
Sodium, mg/day 4,077 ⫾ 323 4,152 ⫾ 329 4,168 ⫾ 368
Sodium, mmol·kg body wt⫺1·day⫺1 2.20 ⫾ 0.03 2.23 ⫾ 0.06 2.24 ⫾ 0.00
Magnesium, mg/day 463 ⫾ 19 447 ⫾ 15 421 ⫾ 26

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Phosphorus, mg/day 1,778 ⫾ 106 1,641 ⫾ 44 1,606 ⫾ 104
Iron, mg/day 13.3 ⫾ 0.6 13.8 ⫾ 0.9 14.5 ⫾ 1.0
Fluoride, ␮g/day 2,893 ⫾ 88 1,597 ⫾ 143 1,318 ⫾ 61
Iodide, ␮g/day 273 ⫾ 19 119 ⫾ 11 189 ⫾ 12
Zinc, mg/day 11.9 ⫾ 0.6 11.3 ⫾ 0.4 15.8 ⫾ 1.2
Copper, ␮g/day 2,059 ⫾ 81 1,821 ⫾ 83 2,022 ⫾ 105
Biotin, ␮g/day 44.4 ⫾ 1.5 82.2 ⫾ 2.4 51.4 ⫾ 2.3
Folic acid, ␮g/day 790 ⫾ 20 605 ⫾ 19 365 ⫾ 12
Niacin equivalent, ␮g/day 35,229 ⫾ 2,943 24,229 ⫾ 1,552 34,294 ⫾ 2,672
Pantothenic acid, mg/day 7.5 ⫾ 0.5 6.0 ⫾ 0.4 5.2 ⫾ 0.3
Retinol equivalent, ␮g/day 1,902 ⫾ 160 2,020 ⫾ 234 812 ⫾ 15
Vit A retinol, mg/day 0.6 ⫾ 0.0 0.3 ⫾ 0.0 0.5 ⫾ 0.0
Vit B1, mg/day 1.4 ⫾ 0.1 2.1 ⫾ 0.2 1.7 ⫾ 0.2
Vit B12, ␮g/day 6.6 ⫾ 0.8 5.0 ⫾ 0.4 5.5 ⫾ 0.5
Vit B2, mg/day 1.8 ⫾ 0.1 1.8 ⫾ 0.1 1.5 ⫾ 0.1
Vit B6, mg/day 2.7 ⫾ 0.2 2.7 ⫾ 0.3 2.8 ⫾ 0.2
Vit C, mg/day 324 ⫾ 22 408 ⫾ 78 98 ⫾ 6
Vit D, IU/day 1,202 ⫾ 19 1,105 ⫾ 3 1,065 ⫾ 2
Vit E, mg/day 19.9 ⫾ 0.6 13.2 ⫾ 0.8 13.0 ⫾ 0.6
Vit K, ␮g/day 384 ⫾ 36 128 ⫾ 90 108 ⫾ 8
Values are means ⫾ SD. BDC, baseline data collection; HDT, head-down tilt; TEE, total energy expenditure; IU, international units.

All data are presented as means ⫾ SD. The level for statistical
significance was set to ␣ ⫽ 0.05, and ␤ was set to 0.2.
RESULTS
Environmental conditions. Environmental parameters in the
bedrooms, measurement rooms, and common spaces of the :
envihab bed rest facility were recorded every minute, 24 h/day
(Fig. 2). On days with ambient atmosphere (baseline data
collection days and 12° HDT with ambient air), the mean CO2
level was 0.04 ⫾ 0.01%, whereas on days with increased
ambient CO2 (intervention day 12° HDT with 0.5% CO2), the
mean CO2 level was 0.48 ⫾ 0.02%.
General health indicators. General health indicator data are
presented in Table 3. Twenty-four-hour pooled urine volume
had a significant main effect of time (P ⫽ 0.005) and was
found to increase from 2,533.8 ⫾ 324.7 ml at baseline data
collection to 3,038.5 ⫾ 506.1 ml during 12° HDT with ambient
air (P ⫽ 0.04) and from 2,671.8 ⫾ 704.3 ml at baseline data
collection to 3,185.2 ⫾ 325.8 ml during 12° HDT ⫹ 0.5% CO2
(P ⫽ 0.03). However, there was no significant main effect of
atmosphere (P ⫽ 0.4). No significant main effects of time or
atmosphere were found for mean arterial pressure (P ⫽ 0.97,
P ⫽ 0.6), systolic blood pressure (P ⫽ 0.5, P ⫽ 0.6), diastolic
blood pressure (P ⫽ 0.5, P ⫽ 0.8), or heart rate (P ⫽ 0.2, P ⫽
0.4). Body temperature, however, was found to have a main
effect of atmosphere (P ⫽ 0.02) but not time (P ⫽ 0.6). Body
weight showed a significant main effect of time (P ⫽ 0.001)
but not atmosphere (P ⫽ 0.97), with a slight decrease in mean
body weight after 19.5 h HDT (P ⫽ 0.02).
Blood parameters. As shown in Table 4, significant main
effects of time were found for WBC (P ⬍ 0.001), LYM (P ⬍
0.001), MON (P ⫽ 0.003), NEU (P ⬍ 0.001), EOS (P ⬍
0.001), HGB (P ⫽ 0.001), HCT (P ⬍ 0.001), RBC (P ⫽
0.004), MCH (P ⫽ 0.003), and RDW (P ⫽ 0.035). Significant
main effects of atmosphere were found for BAS (P ⫽ 0.036),
MCV (P ⫽ 0.045), PLT (P ⫽ 0.002), and MPV (P ⬍ 0.001).
No main effects were found for MCHC.
DISCUSSION
The SPACECOT study demonstrates the feasibility of an
innovative approach to studying the effects of headward fluid
shifting and elevated ambient CO2 on cerebral and ocular
anatomy and physiology. The implemented approach utilized a
steeper HDT angle (12°) than what is typically used (6°), both

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1402 The SPACECOT Study • Marshall-Goebel K et al.

0.5

0.4

Atmospheric CO2 (%)

0.3

Fig. 2. Atmospheric CO2 percentage in the various rooms

within the : envihab bed rest facility as detected by 0.2
wall-mounted CO2 sensors.

0.1

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with and without increased ambient CO2, to decipher the was implemented with a combination of gold-standard and
potential role of atmospheric conditioning during HDT bed novel noninvasive technologies, allowing for a full body sys-
rest. In addition, the study implemented a closely controlled tem physiological analysis. While it is unclear whether HDT
environment and strict adherence to standardization plans and bed rest is a valid model for the ocular changes that occur
the HDT position. A full comprehensive examination of cere- during spaceflight, our approach aimed to investigate the ce-
bral, ocular, and cardiopulmonary physiological parameters rebral and ocular effects of two major hypothesized contribut-
ing factors to the VIIP syndrome: headward fluid shifting and
Table 3. Health indicators were measured at seated baseline exposure to chronically elevated ambient CO2 concentrations.
the morning before going into the HDT position and the next The :envihab facility was able to provide full environmental
morning after being in the 12° HDT position for 19.5 h control and continuous monitoring of atmospheric conditions,
light intensity and spectrum parameters, temperature, and hu-
Condition Baseline 19.5 h HDT midity. Atmospheric condition during HDT had little or no
Body Weight, kg additional effect on daily blood pressure, heart rate, urine
HDT ⫹ 0.5% CO2 82.03 ⫾ 7.71 81.33 ⫾ 7.36* output, body weight, or blood count parameters. However,
HDT ⫹ amb 82.1 ⫾ 7.93 81.42 ⫾ 7.56 HDT affected many of the parameters, regardless of atmo-
Body Temperature, °C spheric condition, notably inducing increased urine output,
HDT ⫹ 0.5% CO2 35.25 ⫾ 0.93 35.27 ⫾ 0.98
HDT ⫹ amb 34.88 ⫾ 0.86 34.88 ⫾ 0.86
WBC, NEU, HCT, and HGB. Diuresis is a known response to
return the perceived HDT-induced increases in central blood
Mean Arterial BP, mmHg
HDT ⫹ 0.5% CO2 86.4 ⫾ 4.77 85.32 ⫾ 5.04 volume close to baseline values (2). Increased HCT and HGB
HDT ⫹ amb 85.88 ⫾ 6.66 86.58 ⫾ 5.93 are also likely due to the shift in central blood volume.
Systolic BP, mmHg Notably, WBC count increased with HDT and was further
HDT ⫹ 0.5% CO2 113.33 ⫾ 3.2 113.85 ⫾ 5.86 aggravated by the short duration exposure to 3% CO2. The
HDT ⫹ amb 112.88 ⫾ 8.86 117.05 ⫾ 5.74 percentage share of LYM and granulocytes was shifted to NEU
Diastolic BP, mmHg in HDT, and when translated into absolute counts, there is little
HDT ⫹ 0.5% CO2 72.93 ⫾ 6.72 71.05 ⫾ 5.95
HDT ⫹ amb 72.38 ⫾ 6.94 71.32 ⫾ 7.72
change in LYM count while innate immune cells are increased.
Heart Rate, beats/min
The observed increase in polymorphonuclear cells in our study
HDT ⫹ 0.5% CO2 60.5 ⫾ 6.82 61.22 ⫾ 9.92 is consistent with an acute stress response. Typically, physio-
HDT ⫹ amb 62.88 ⫾ 8.51 59.05 ⫾ 8.85 logical stress leads to demargination of NEU from the blood
Values are means ⫾ SD. BP, blood pressure; amb, ambient air. *Significant
vessel wall into the lumen of the blood, where they enter the
difference from respective baseline measures from post hoc Bonferroni testing systemic circulation and are thus measurable. This is consistent
(P ⬍ 0.05). with a previous HDT study in which eight volunteers under-

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 The SPACECOT Study • Marshall-Goebel K et al. 1403
Table 4. Blood count parameters at baseline and during the two HDT campaigns
BDC (Morning of HDT) 4.5 h HDT 24 h HDT 28.5 h HDT ⫹ 3% CO2

White Blood Cells, ⫻ 10 /␮l

3

HDT ⫹ 0.5% CO2 5.67 ⫾ 1.37 6.67 ⫾ 1.63 7 ⫾ 1.79* 8.17 ⫾ 2.32‡
HDT ⫹ amb 6 ⫾ 1.26 7.17 ⫾ 2.23 7.17 ⫾ 1.47 7.83 ⫾ 2.14‡
Lymphocytes, %
HDT ⫹ 0.5% CO2 42.17 ⫾ 4.79 35.17 ⫾ 5.15‡ 33 ⫾ 4.29‡ 30.17 ⫾ 5.64‡
HDT ⫹ amb 42.83 ⫾ 3.43 36 ⫾ 5.9‡ 32.33 ⫾ 5.16‡ 31.17 ⫾ 6.65‡
Monocytes, %
HDT ⫹ 0.5% CO2 10.67 ⫾ 1.63 10.5 ⫾ 1.05 11.33 ⫾ 1.86 11.67 ⫾ 1.37
HDT ⫹ amb 11.17 ⫾ 1.33 10 ⫾ 1.79 10.83 ⫾ 0.98 10.83 ⫾ 1.17
Neutrophils, %
HDT ⫹ 0.5% CO2 43.67 ⫾ 5.13 51.67 ⫾ 5.32‡ 53.5 ⫾ 4.76‡ 55.83 ⫾ 6.82‡
HDT ⫹ amb 42.83 ⫾ 4.12 51.5 ⫾ 5.96‡ 54.33 ⫾ 5.24‡ 55.33 ⫾ 6.22‡
Basophils, %
HDT ⫹ 0.5% CO2 0⫾0 0⫾0 0.17 ⫾ 0.41 0.33 ⫾ 0.52
HDT ⫹ amb 0.5 ⫾ 0.55 0.17 ⫾ 0.41 0.17 ⫾ 0.41 0.17 ⫾ 0.41
Eosinophils, %
HDT ⫹ 0.5% CO2 2.83 ⫾ 0.98 2.17 ⫾ 0.75 2 ⫾ 0.89 1.83 ⫾ 0.75*

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HDT ⫹ amb 3.33 ⫾ 0.82 2.33 ⫾ 0.52* 2.17 ⫾ 0.75† 2 ⫾ 0.89‡
Hemoglobin, g/dl
HDT ⫹ 0.5% CO2 15 ⫾ 0.89 15.17 ⫾ 1.17 15.5 ⫾ 0.84 15.5 ⫾ 0.84
HDT ⫹ amb 14.83 ⫾ 1.17 15.33 ⫾ 1.03 15.5 ⫾ 1.05 15.83 ⫾ 1.17†
Hematocrit, %
HDT ⫹ 0.5% CO2 43 ⫾ 2.76 43.17 ⫾ 3.31 44.5 ⫾ 3.02 45 ⫾ 2.19
HDT ⫹ amb 42.67 ⫾ 3.39 43.5 ⫾ 3.08 44.83 ⫾ 2.99* 45.5 ⫾ 2.35†
Red Blood Cells, ⫻ 106/␮l
HDT ⫹ 0.5% CO2 5⫾0 5⫾0 5.33 ⫾ 0.52 5.33 ⫾ 0.52
HDT ⫹ amb 5⫾0 5⫾0 5.17 ⫾ 0.41 5.33 ⫾ 0.52
Mean Corpuscular Volume, fl
HDT ⫹ 0.5% CO2 85.67 ⫾ 4.5 85.33 ⫾ 4.18 85.67 ⫾ 4.5 85.67 ⫾ 3.93
HDT ⫹ amb 85.67 ⫾ 4.5 86 ⫾ 4.52 86 ⫾ 4.52 85.83 ⫾ 4.22
Mean Corpuscular Hemoglobin, pg
HDT ⫹ 0.5% CO2 29.33 ⫾ 1.63 29.83 ⫾ 1.47 30 ⫾ 1.41† 29.83 ⫾ 1.47
HDT ⫹ amb 29.67 ⫾ 1.37 30 ⫾ 1.41 30 ⫾ 1.41 29.83 ⫾ 1.47
Mean Corpuscular Hemoglobin Concentration, g/dl
HDT ⫹ 0.5% CO2 34.5 ⫾ 0.55 34.67 ⫾ 0.52 34.67 ⫾ 0.52 34.83 ⫾ 0.41
HDT ⫹ amb 34.67 ⫾ 0.52 35 ⫾ 0.63 34.67 ⫾ 0.52 34.83 ⫾ 0.41
Red Blood Cell Distribution Width, %
HDT ⫹ 0.5% CO2 12.67 ⫾ 0.52 12.5 ⫾ 0.84 12.5 ⫾ 0.84 12.33 ⫾ 0.82
HDT ⫹ amb 12.67 ⫾ 1.03 12.5 ⫾ 1.05 12.5 ⫾ 1.05 12.33 ⫾ 0.82
Platelets, ⫻ 103/␮l
HDT ⫹ 0.5% CO2 224.33 ⫾ 41.25 231.33 ⫾ 42.87 228.17 ⫾ 42.93 233.5 ⫾ 44.23
HDT ⫹ amb 221.67 ⫾ 35.64 245 ⫾ 24.9 245.17 ⫾ 22.12 252.67 ⫾ 32.89
Mean Platelet Volume, fl
HDT ⫹ 0.5% CO2 8.83 ⫾ 1.17 8.5 ⫾ 1.05 8.33 ⫾ 1.03 8.17 ⫾ 1.17
HDT ⫹ amb 8.17 ⫾ 0.75 7.83 ⫾ 0.98 8 ⫾ 0.89 8 ⫾ 0.89
Values are means ⫾ SD. *P ⬍ 0.05, †P ⬍ 0.01, ‡P ⬍ 0.001, change from respective baseline measures derived from post hoc Bonferroni testing.

went 6° HDT for 42 days and polymorphonuclear cells in-
creased during HDT, whereas the T-lymphocytes and mono-
cytes did not change (12).
In contrast to the slight elevation of WBC count, the vital
signs including blood pressure did not significantly change
from baseline to the late HDT time point (Table 3) in either the
HDT alone or HDT with 0.5% CO2 condition. This is consis-
tent with findings from previous bed rest studies (10, 13), and
there may be several explanations for this. First, the subjects
may have adapted to the stress of the environment and, there-
fore, the blood pressure remained unchanged from baseline.
Second, a decrease in plasma volume related to HDT-induced
diuresis may have occurred, which would lower the central
venous pressure and preload, thus reducing cardiac output
given a stable heart rate.
Nutritional intake was highly standardized and regulated in
the presented study and is important to take into consideration
when interpreting results of other physiological systems, be-
cause diet may affect multiple organ system function including
cardiovascular and cerebrovascular parameters. Notably, so-
dium content was set to ~4 g/day in this study, standard for bed
rest studies, and is an important factor to monitor closely,
because sodium intake could have effects on blood pressure
and fluid retention. Whether sodium intake has an effect on
cerebral and ocular changes in spaceflight is unknown; however,
if vascular fluid content and thus volume increases, this may also
have effects on the cerebrovascular system. Given that ICP is
related to cerebral venous outflow and central venous pressure, it
is possible that a high sodium diet may contribute to a higher
circulating venous volume and affect ICP.

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1404 The SPACECOT Study • Marshall-Goebel K et al.
The unique platform of the SPACECOT study with a steeper
degree of HDT as well as atmospheric conditioning is consid-
ered to be more challenging for subjects than standard HDT
bed rest studies, and, therefore, new procedures were imple-
mented to ensure both subject comfortability and successful
completion of the study. First, we selected several subjects
with prior bed rest experience who were mentally prepared for
the bed rest experience. Furthermore, we extended subject
recruitment to include brief HDT exposure for both mental and
physical familiarization with the demands of bed rest at a
steeper HDT angle, as well as an MRI session to exclude
unknown anxiety disorders or claustrophobia. In addition, a
full day of familiarization was added before the first day of
baseline data collection, to introduce the subjects to all of the
study measurement techniques and the busy schedule. This is
thought to have alleviated some of the stress associated with
the complex procedures of the study, allowing subjects to be
more relaxed and comfortable during actual baseline measure-
ments the following day. Furthermore, pillows are normally
provided to subjects in bed rest studies; however, the use of a
pillow places the head and eyes slightly above the intended
degree of full body tilt. Thus, pillows were not allowed in the
SPACECOT study, an important consideration for future bed
rest studies focused on cerebral and ocular physiology. In
addition, bed rest studies often involve procedures that require
subjects to move into the supine position (e.g., MRI scans and
exercise regimes). In the present study, we made every effort to
ensure the 12° HDT position was maintained during the entire
bed rest period. This included having dedicated subject mon-
itors assigned to every subject to ensure subject safety and
comfort as well as maintain head, neck, and body position at
12° HDT during bed rest, transfers to other beds, and meal
times. In addition, custom-built wedges at 12° HDT were
implemented into the MRI scanner to maintain the HDT
position. Furthermore, a mask and tank system was used when
subjects were required to leave the bed rest facility for all
conditions (ambient and increased CO2) to retain the blinded
aspect of the study and atmospheric conditioning when in the
MRI scanner. It is important to note that subjects were unlikely
to be in a fully relaxed state due to the stressful environment of
the busy study. However, this is thought to be similar to the
stressful environment that astronauts may endure during space-
flight.
With the implementation of a steeper HDT position to
investigate the effects of cephalad fluid shifting, we determined
inherent limitations and hereby provide recommendations for
successful future implementation of this approach. First, the
12° HDT position can lead to subjects slowly slipping down
the bed. We therefore lined the mattress with a nonskid pad to
prevent slippage. In addition, lower back pain was a common
complaint, and countermeasures against this should be in place,
including heating pads and daily physical therapy massages
applied to the lower back. Given the relatively short duration of
the exposure to 12° HDT, we cannot extrapolate as to whether
the back pain would have resolved over time; however, back
pain also frequently occurs with a milder degree of HDT and
usually resolves spontaneously after adaptation. Finally, eating
was found to be more difficult at this steeper HDT angle, and
therefore dedicated subject monitors also assisted subjects with
eating, and the menu was adapted to provide more finger food
such as sandwiches. Adverse events of the study included one
J Appl Physiol • doi:10.1152/japplphysiol.00885.2016 • www.jappl.org
episode of emesis, and one subject was unable to urinate in the
12° HDT position during the first campaign and had to have a
urinary catheter inserted. In the subsequent campaign, this
subject was briefly moved to the 0° position for urination. Data
were examined to determine whether measurements obtained
from this one subject were outside of one standard deviation of
the group mean, and this was determined not to be the case.
With this one exception, in general, the subjects were able to
urinate and defecate at the 12° HDT position without difficulty.
There are important differences between real microgravity
and HDT bed rest that must be taken into account when
interpreting results, most notably, the presence of gravitational
vectors in the latter. Unlike in microgravity where all hydro-
static gradients are abolished, a small foot-to-head vector is
created during HDT to create a headward fluid shift similar to
what may be experienced in microgravity. In addition, a Gx
(chest to back) gravitational vector is present in HDT bed rest,
which may result in different cardiovascular and pulmonary
physiological responses compared to microgravity. Further-
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more, this study aimed to investigate the immediate effects of
headward fluid shifting in the presence of elevated ambient
CO2 to understand potential mechanistic underlying factors
that, if persistent, could lead to ophthalmic changes associated
with the VIIP syndrome. However, because ophthalmic changes
in astronauts appear after months of microgravity exposure, we
cannot assume that observed changes can be directly applied to
longer term HDT bed rest or microgravity.
To our knowledge, this is the first study to implement a
steeper HDT position with increased ambient CO2 for more
than 24 h of bed rest in healthy human subjects as an approach
to investigate two hypothesized contributing factors to ocular
changes in astronauts: headward fluid shifting as well as
exposure to chronically elevated ambient CO2. Further work is
needed to determine whether a longer duration of bed rest
within this model is well tolerated and leads to physiological
and anatomical changes similar to those associated with space-
flight.
ACKNOWLEDGMENTS
We acknowledge the SPACECOT investigators group (in alphabetical
order): Mathias Basner, Christine Becker, Eusebia Calvillo, Jonathan Clark,
Rahul Damani, Wolfgang Doering, Christian Dohmen, Peter Gauger, Darius
Gerlach, Olga Hand, Khader Hasan, Elfriede Huth, Bernd Johannes, Larry
Kramer, Gabriele Kraus, Uwe Mittag, Klaus Muller, Jad Nasrini, Ben Nieder-
berger, Dirk Poddig, Matthias Putzke, Martina Sagner, Haleh Sangi-Haghpey-
kar, Irmtrud Schrage-Knoll, Wolfram Sies, Claudia Stern, Henning Stetefeld,
Brian Stevens, Annette von Waechter, Tobias Weber, and Martin Wittkowski.
We also thank the subjects for their time and steadfast dedication. We
appreciate the technical support and advice provided by Mark Shusterman,
Mitch Levinson, Rolandas Zaeklis, Adi Tsalach, Avihai Ron, and Tracy
Johnson. Finally, this study would not have been possible without the encour-
agement and support of Rupert Gerzer, Julie Do, and Jeff Sutton.
GRANTS
This study was supported by the National Space Biomedical Research
Institute through NASA NCC 9 –58, the Baylor College of Medicine Center for
Space Medicine, and the German Aerospace Center Institute of Aerospace
Medicine.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
K.M.-G., E.M., D.D., G.S., J.I.S., C.V.R., P.F.-M., U.L., J.R., and E.M.B.
conceived and designed research; K.M.-G., E.M., D.D., G.S., J.I.S., C.V.R.,
 The SPACECOT Study • Marshall-Goebel K et al.
P.F.-M., U.L., J.R., and E.M.B. performed experiments; K.M.-G., E.M.,
P.F.-M., and E.M.B. analyzed data; K.M.-G., E.M., U.L., and E.M.B. inter-
preted results of experiments; K.M.-G. and J.R. prepared figures; K.M.-G.
drafted manuscript; K.M.-G., E.M., D.D., G.S., J.I.S., C.V.R., P.F.-M., U.L.,
J.R., and E.M.B. edited and revised manuscript; K.M.-G., E.M., D.D., G.S.,
J.I.S., C.V.R., P.F.-M., U.L., J.R., and E.M.B. approved final version of
manuscript.
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