Impact of circadian misalignment on energy
metabolism during simulated nightshift work
Andrew W. McHilla,1, Edward L. Melansonb,c, Janine Higginsd, Elizabeth Connicke, Thomas M. Moehlmana,
Ellen R. Stotharda, and Kenneth P. Wright Jr.a,b,2
a
Sleep and Chronobiology Laboratory, Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO 80309; and Divisions of
b
Endocrinology, Metabolism, and Diabetes, cGeriatric Medicine, and eInfectious Diseases and dDepartment of Pediatrics, University of Colorado Anschutz
Medical Campus, Aurora, CO 80045
Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 21, 2014
(received for review June 25, 2014)
Eating at a time when the internal circadian clock promotes sleep
is a novel risk factor for weight gain and obesity, yet little is
known about mechanisms by which circadian misalignment leads
to metabolic dysregulation in humans. We studied 14 adults in a
6-d inpatient simulated shiftwork protocol and quantified changes
in energy expenditure, macronutrient utilization, appetitive hor-
mones, sleep, and circadian phase during day versus nightshift
work. We found that total daily energy expenditure increased by
4% on the transition day to the first nightshift, which consisted
of an afternoon nap and extended wakefulness, whereas total
daily energy expenditure decreased by 3% on each of the second
and third nightshift days, which consisted of daytime sleep fol-
lowed by afternoon and nighttime wakefulness. Contrary to
expectations, energy expenditure decreased by 1216% during
scheduled daytime sleep opportunities despite disturbed sleep.
The thermic effect of feeding also decreased in response to a late
dinner on the first nightshift. Total daily fat utilization increased
on the first and second nightshift days, contrary to expectations,
and carbohydrate and protein utilization were reduced on the
second nightshift day. Ratings of hunger were decreased during
nightshift days despite decreases in 24-h levels of the satiety hor-
mones leptin and peptide-YY. Findings suggest that reduced total
daily energy expenditure during nightshift schedules and reduced
energy expenditure in response to dinner represent contributing
mechanisms by which humans working and eating during the bi-
ological night, when the circadian clock is promoting sleep, may
increase the risk of weight gain and obesity.
|
insufficient sleep melatonin | diet-induced thermogenesis |
|
eating at night appetite
E merging evidence from nonhuman animal models indicates
a fundamental interplay between circadian and metabolic
physiology (1, 2) with implications for health and disease (35).
Eating at inappropriate circadian times (e.g., at night) is con-
sidered a novel risk factor for weight gain and obesity, yet little
research has been conducted in humans on this topic. The cir-
cadian time-keeping system in humans modulates energy me-
tabolism so that wakefulness, activity, and food intake are
promoted during the solar day and sleep, inactivity, and fasting
occur during the solar night (2). With the widespread use of
electrical lighting, however, work and social activities are capable
of being extended further into the night (6, 7). Being awake during
the biological night leads to disturbed physiology and behavior,
because it creates a state of desynchrony between the circadian
clock and wakefulnesssleep cycle known as circadian mis-
alignment. Circadian misalignment is common in shiftwork. More
than 20% of adults in the United States work nontraditional hours
(8) and eat some of their meals during the biological night (9),
which can increase blood glucose and triacylglycerol levels in re-
sponse to a high-carbohydrate versus high-fat diet (10) and in-
crease low-density lipoprotein cholesterol levels (11). Further,
1730217307 | PNAS | December 2, 2014 | vol. 111 | no. 48
shiftwork is associated with increased risk for obesity (12), meta-
bolic syndrome (13), and diabetes (14).
Evidence from nonhuman animal models reveals that con-
sumption of calories at inappropriate circadian times contributes
to weight gain (15, 16). Further, caloric intake during typical
sleep time leads to greater weight gain than the same caloric
intake during typical waketime (15, 16). Mechanisms by which
eating during the biological night increases risk of weight gain in
humans remain unknown. Therefore, the primary aim of this
study was to examine the influence of circadian misalignment on
total daily energy expenditure (EE), EE after food intake (re-
ferred to as the thermic effect of food; TEF), and total daily
macronutrient utilization (use of energy from fats, carbohy-
drates, and proteins) in humans via whole-room indirect calo-
rimetry. The 6-d inpatient nightshift work simulation protocol
was also used to examine the influence of circadian misalignment
on sleep staging, EE during scheduled sleep and for sleep stages,
hunger ratings, and the appetitive hormone ghrelin and satiety
hormones leptin and peptide-YY (PYY). We hypothesized that
circadian misalignment would disrupt sleep, reduce fat utiliza-
tion (because carbohydrate utilization is tightly controlled in
humans) (17), reduce TEF following food intake during the bi-
ological night, and alter circulating concentrations of appetitive
hormones associated with increased appetite [increase ghrelin
and reduce leptin (5, 18) and PYY levels]. We also hypothesized
Significance
Demands of modern society force many work operations into
the night, when the intrinsic circadian timing system promotes
sleep. Overnight shiftwork is associated with increased risk for
adverse metabolic health and sleep disruption. Uncovering po-
tential physiological mechanisms that contribute to metabolic
dysregulation when work and eating occur at inappropriate
circadian times is vital to the development of effective treatment
strategies. In this study, healthy volunteers underwent a com-
monly used simulated shiftwork protocol to quantify changes in
metabolic, sleep, and circadian physiology when working and
eating during the night as compared with a traditional day work
schedule. We demonstrate that nightshift work reduces total
daily energy expenditure, representing a contributing mecha-
nism for unwanted weight gain and obesity.
Author contributions: A.W.M., E.L.M., J.H., and K.P.W. designed research; A.W.M., E.L.M.,
J.H., E.C., T.M.M., E.R.S., and K.P.W. performed research; A.W.M., E.L.M., J.H., T.M.M., E.R.S.,
and K.P.W. analyzed data; and A.W.M., E.L.M., J.H., E.C., T.M.M., E.R.S., and K.P.W. wrote the
paper.
Conflict of interest statement: There are no conflicts of interest directly related to this
project. E.L.M. and K.P.W. have current funding through a Philips research grant.
This article is a PNAS Direct Submission.
1
Division of Sleep and Circadian Disorders, Department of Medicine, Brigham and Wom-
ens Hospital and Division of Sleep Medicine, Harvard Medical School, Boston, MA 02115.
2
To whom correspondence should be addressed. Email: kenneth.wright@colorado.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1412021111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1412021111
that circadian misalignment would increase EE during scheduled
daytime sleep opportunities due to increased arousals (19), but 7.5

Energy Expenditure (kjoules/min)

that a hypothesis can also be made for decreased total daily EE (9). $

#
1.8
$

Energy Expenditure (kcal/min)

# *
7.0 # 1.7
Results
*
# # # # *
##
* # * * * #
Circadian Melatonin Rhythm and Sleep Staging. Circadian mis-
6.5
#
* ** *# *
1.5
alignment occurred during simulated nightshift work (Fig. 1). 6.0 # #
* *
# # 1.4
Fig. S1 shows that the circadian melatonin rhythm did not adapt
to the nightshift schedule, and thus sleep occurred during the 5.5 1.3
biological day when melatonin levels were low and wakefulness
occurred during the biological night when melatonin levels were 5.0 1.2
high. Total sleep time, sleep efficiency, and percentage and 4.5 1.1
minutes of stage 2 sleep were lower, minutes of wakefulness after Day 2- Baseline
Day 3- Nightshift 1
sleep onset (WASO) was higher, and average duration of awak- 4.0 Day 4- Nightshift 2 1.0
enings was increased during daytime sleep (Table S1). Further, Nap Day 5- Nightshift 3
daytime sleep opportunities showed shorter latencies to sleep 3.5 Dayme Sleep Habitual Night 0.8
onset (SOL), persistent sleep (LPS), and rapid eye movement 09 13 17 21 01 09
05
sleep (REML). Last, percentage and minutes of REM sleep were
lower during daytime sleep before nightshift 2 versus baseline. Relative Clock Hour
Fig. 2. Hourly energy expenditure across the simulated shiftwork protocol
Energy Expenditure, Macronutrient Utilization and Balance, and (n = 14; P values, planned comparisons for day, two-tailed). The gray line
Thermic Effect of Food. Regardless of when sleep occurred, aver- represents day 2 (baseline), the black line is day 3 (nightshift 1), the blue line
age hourly EE was lower during scheduled sleep than wakeful- is day 4 (nightshift 2), and the red line is day 5 (nightshift 3). Black boxes
ness (Fig. 2). EE during the 2-h daytime nap (5.1 0.7 kJ/min; along the x axis indicate scheduled sleep opportunities (day 2 nighttime, day
1.2 0.02 kcal/min SEM) was reduced to similar levels seen in 3 nap, days 45 daytime). *Differences between baseline and nightshift 1;
the first 2 h of the baseline nighttime sleep opportunity (Fig. 2). #
differences between baseline and nightshift 2; differences between base-
Compared with baseline (day 2), total daily EE was 353 41.8 kJ line and nightshift 3; differences between nightshifts 1 and 2; differences
(84 10 kcal) higher on nightshift 1 (day 3), which included between nightshifts 1 and 3; $differences between nightshifts 2 and 3; P <
the 2-h nap and extended wakefulness, and was 217 69.9 kJ 0.05. Error bars are SEM.
(52 16.7 kcal) and 247 66.2 kJ (59 15.8 kcal) lower on
nightshifts 2 and 3 (days 4 and 5), respectively (P < 0.025; Fig.
3A). Average per minute EE during scheduled wakefulness was nightshift 1 (0.7 1.3%; P < 0.025) versus baseline (4.8 1.0%) and
2.5% less on nightshift 1 (day 3) versus other days (P < 0.025; nightshift 3 (5.0 0.9%). TEF postdinner on nightshift 2 was in-
Fig. 3B) but was similar between baseline and nightshifts 2 and 3 termediate (2.8 0.7%) and not significantly different from
(P > 0.33). Post hoc analyses showed that average per minute EE other days.
during scheduled daytime sleep was 12% less before nightshifts
2 and 3 versus baseline (Fig. 3C). SleepWakefulness Stage and Energy Expenditure. EE did not sig-
Subjects were given meals designed to meet energy balance needs nificantly differ between stages of consolidated sleep within sleep
at baseline (7,643 1,083 kJ/d; 1,827 259 kcal/d). Table 1 shows opportunities (all P > 0.10; Fig. S2 and Table S2). During the
that total daily fat utilization was 8% higher on nightshift 1 and baseline sleep opportunity, EE was higher during wakefulness
18% higher on nightshift 2 versus baseline (post hoc), resulting in prior to sleep onset (WPSO; from lights out to LPS) and WASO

PHYSIOLOGY
negative fat balance for all subjects on the first two shiftwork days. than during stage 2 and REM sleep. Further, EE was higher
Furthermore, total daily carbohydrate utilization was 20% lower during WPSO than stage 1 and slow-wave sleep (SWS) (P < 0.05;
on nightshift 2 versus baseline, resulting in greater positive carbo- Table S2). During daytime sleep opportunities, however, EE
hydrate balance. Also, protein utilization was 10% lower on during WPSO was lower than that during consolidated sleep
nightshift 2 versus baseline. Macronutrient utilization on nightshift stages, whereas EE during WASO was higher than most con-
3 was similar to baseline (Table 1). Postdinner TEF was lower on solidated sleep stages and WPSO (P < 0.05; Table S2).
EE was lower during WPSO, WASO, stage 2, and SWS for the
daytime versus baseline nighttime sleep opportunity (P < 0.05;
Fig. S2). Further, EE was lower during stage 1 sleep for daytime
Relative Clock Hour sleep before nightshift 2 and during REM sleep for daytime
8 16 24 8 sleep before nightshift 3, both versus baseline. When examining
1 D S differences in EE between baseline and daytime sleep opportu-
nities averaging across all scored sleep epochs, subjects expended
Day of Study

2 B L D S
3 B L S D S
4 B L D S 1.14 0.002 kcal/min P < 0.001), respectively.
5 B L D S Appetitive Hormones and Hunger Ratings. Average 24-h ghrelin
Fig. 1. Study protocol. Open bars represent room light (<40 lx). Black bars
represent scheduled polysomnography-recorded sleep. Time of day is plot-
ted as relative clock hour with scheduled waketime arbitrarily assigned a
value of 0800 hours and all other times referenced to this value. Shaded
areas indicate scheduled wakefulness in dim light (<1 lx). B, breakfast; D,
dinner; L, lunch; S, snack. Subjects were allowed three brief scheduled breaks
per day to stand up and retrieve meals and use the toilet for bowel move-
ments <3 m from the bed. Outside of scheduled breaks, subjects were given
a urinal or bedpan when needed. Wakefulness and subject compliance
during the constant posture protocol were verified via continuous moni-
toring by research staff and electroencephalography (EEG) recordings.
16% and 12% less energy during sleep before nightshifts 2
(4.0 0.02 kJ/min; 0.96 0.005 kcal/min) and 3 (4.2 0.02 kJ/
min; 1 0.005 kcal/min) versus baseline (4.8 0.01 kJ/min;
levels were similar across days (Fig. 4A), whereas average 24-h
leptin levels decreased by 41% on nightshift 2 versus baseline
and by 33% and 35% versus nightshifts 1 and 3, respectively
(Fig. 4B). Similarly, average 24-h PYY levels decreased by 11%
on nightshift 2 versus baseline and by 7% and 13% versus
nightshifts 1 and 3, respectively (Fig. 4C). In addition, average
24-h PYY levels increased by 6% on nightshift 3 versus
nightshift 1. Fig. S3 shows that daily patterns of ghrelin, leptin,
and PYY predominantly follow the sleepwakefulness fasting
feeding cycle. Hunger ratings decreased across days and were
21% and 35% lower on nightshifts 2 and 3, respectively, versus

McHill et al. PNAS | December 2, 2014 | vol. 111 | no. 48 | 17303

 A 10000 2381 nightshift days. Further, carbohydrate and protein utilization de-
creased on the second nightshift day. Last, subjects exhibited de-
Energy Expenditure (kjoules/day)

Energy Expenditure (kcal/day)

creased hunger ratings despite similar levels of the hunger-
stimulating hormone ghrelin and decreased levels of the satiety
9000 2143 hormones leptin and PYY during the nightshift schedule. We
studied lean, healthy subjects and strictly controlled for food in-
take, activity, posture, and light exposure so that we could examine
the influence of circadian misalignment per se on energy metab-
8000 1905 olism. Our control over food intake and activity does not permit us
to make strong comments about how total daily EE, energy bal-
ance, and hunger may change during uncontrolled conditions,
something that should be examined in future studies. Also, we did
7000 1666
Baseline Nightshift 1 Nightshift 2 Nightshift 3
not measure for possible changes in body composition nor did we
Transition have a control group without circadian misalignment; however,
Total Daily longer time in the laboratory with insufficient sleep induces con-
sistent increases in 24-h EE (20). Future studies are needed to
B 7.5 1.8
Energy Expenditure (kjoules/min)

determine whether other subject populations and behavioral/

Energy Expenditure (kcal/min)

7.0 1.7 environmental factors, including other models of circadian mis-
6.5 1.5 alignment (e.g., rotating shift work, additional night shifts, circa-
dian sleepwake disorders), influence metabolic physiology. Some
6.0 1.4 findings that were in opposite directions than expected require
5.5 1.3 replication.
5.0 1.2
Total Daily Energy Expenditure. Using a common nightshift schedule
4.5 1.1 simulation, we found that acute circadian misalignment increased
4.0 1.0
total daily EE on the transition day to nightshift 1 and decreased
total daily EE on nightshifts 2 and 3. Increased total daily EE on
3.5 0.8 the transition day was expected due to increased time awake and is
Nightshift 1
Baseline Nightshift 2 Nightshift 3
Transition consistent with prior findings from our laboratory that sleep
Scheduled Wakefulness deprivation increases energy expenditure (19, 20). Decreased EE
per minute during scheduled wakefulness on nightshift 1, however,
C 7.5
ph< 0.025
1.8 indicates that the duration of wakefulness, and not an increase in
Energy Expenditure (kjoules/min)

Energy Expenditure (kcal/min)

7.0
ph< 0.025
6.5
6.0
5.5
5.0
4.5
4.0
3.5
Baseline
Prior to Nightshift 2 Prior to Nightshift 3
Scheduled Sleep
Fig. 3. Total daily (24-h) (A) and scheduled wakefulness (B) and sleep (C)
energy expenditure (n = 14; P values, planned comparisons for day, two-
tailed). Solid lines represent significant differences at the end of each line
(modified Bonferroni, P < 0.025). Exploratory post hoc (ph) two-tailed P
values are shown for scheduled sleep EE, as findings were in the opposite
direction of that hypothesized. Error bars are SEM.
baseline and were 28% and 18% lower on nightshift 3 versus
nightshifts 1 and 2, respectively (Fig. 4D). A nonsignificant trend
occurred for lower hunger ratings on nightshift 2 versus night-
shift 1 (Fig. 4D).
Discussion
Findings from nonhuman animal models indicate that food in-
take during typical sleep time leads to metabolic dysregulation.
We show that total daily EE was significantly increased on the
first nightshift day due to extended wakefulness and was de-
creased on the second and third nightshift days compared with
baseline. Additionally, decreases in total daily EE during the
nightshift schedule were driven predominantly by decreases in
sleeping EE and the EE of wakefulness during scheduled sleep
opportunities. We also show that the TEF in response to dinner
decreased on the first nightshift compared with baseline. Un-
expectedly, fat utilization increased on the first and second
1.7 the metabolic cost of such wakefulness, is responsible for in-
creased total daily EE. Our findings of decreased total daily EE
1.5
during subsequent nightshift days may represent a contributing
1.4 mechanism for unwanted weight gain observed in shiftworkers.
1.3
Because shiftworkers report eating similar total 24-h calories as
daytime workers (9, 11), the 217 to 247 kJ/d (5259 kcal/d)
1.2 reduction in total daily EE we observed could lead to positive
1.1
energy balance and weight gain over time (21).
TEF accounts for 10% of total daily EE and reflects the
1.0 energy needed for metabolism of consumed calories. A reduced
0.8 TEF could contribute to reductions in 24-h EE and, if food in-
Daytime Sleep Daytime Sleep take remained stable, weight gain could ensue. We found that
the TEF after a late dinner on nightshift 1 was lower compared
with food consumed at a typical dinnertime. This decreased TEF
may represent a contributing mechanism underlying the reported
risk of weight gain associated with evening food intake. For ex-
ample, food consumed past 2000 hours predicts higher body
mass index (BMI) when controlling for sleep duration and timing
(22), and night eating syndrome, a disorder in which individuals
consume 25% of calories after their evening meal (23), is as-
sociated with a higher BMI (24). Reduced TEF after the late
dinner is consistent with prior findings of an acute decrease in
TEF after a meal consumed at 0100 hours compared with 0900
hours or 1700 hours (25). Our finding that TEF in response to
dinner was intermediate on nightshift 2 and returned to levels
similar to baseline on nightshift 3 was unexpected, but may
suggest physiological adaptation in TEF during the acute chal-
lenge posed by such nightshift schedules.
Macronutrient Utilization and Balance. Higher fat utilization (en-
ergy used from fat) on nightshifts 1 and 2 was not expected. This
finding and the negative fat balance (fat loss), lower carbohy-
drate and protein utilization (energy used from carbohydrates
and protein), and higher positive carbohydrate balance (carbo-
hydrate storage) may represent acute physiological responses to
higher EE during sleep deprivation, especially as the food intake
provided to subjects was the amount predicted to maintain en-
ergy balance at baseline and thus subjects were in negative en-
ergy balance (weight loss) during sleep deprivation.

17304 | www.pnas.org/cgi/doi/10.1073/pnas.1412021111 McHill et al.

 Table 1. Total daily, macronutrient utilization and balance for baseline and nightshift days
Day 2: baseline
Measure, g/d (n = 14)
Macronutrient utilization
Carbohydrate 210.0 (8.1)
Fat 84.1 (6.6)
Protein 58.8 (3.0)
Macronutrient balance
Carbohydrate 26.5 (10.9)
Fat 21.6 (5.4)
Protein 11.5 (2.8)
Values in parentheses are SEM.
*Differences between baseline and nightshift 2.
Differences between nightshift 1 and nightshift 2.
Differences between nightshift 2 and nightshift 3.
Differences between baseline and nightshift 1.
{
Exploratory post hoc differences for fat utilization (day 4 vs. day 2, P = 0.001; day 3, P = 0.01; day 5, P = 0.000005).
Sleep Staging and Energy Expenditure. Consistent with prior find-
ings, we observed that sleep during the daytime was disturbed
(26, 27). Shorter sleep latencies during the nightshift schedule
were expected due to increased sleep drive and the circadian
promotion of sleep (28, 29). Reductions in total sleep time and
stage 2 sleep and increases in duration of arousals were also
expected due to the circadian promotion of arousal during the
latter portion of the daytime sleep opportunity (28). The shorter
REML during daytime sleep is consistent with the circadian
rhythm in REM propensity, as these sleep opportunities were
scheduled near the peak circadian time for REM (28).
Contrary to our hypothesis, we found decreased EE during
scheduled daytime sleep opportunities. However, our findings of
higher EE during awakenings from sleep compared with con-
solidated sleep and of similar EE among consolidated sleep stages
within the sleep opportunity are consistent with prior findings (19,
30). The finding of lower WPSO EE than consolidated sleep stages
for daytime sleep opportunities is contrary to the EE observed at
baseline during the transition from wakefulness to sleep and is
inconsistent with findings from prior research for nighttime sleep
opportunities (19). Decreased EE during sleep and wakefulness of
the daytime sleep opportunities may function to conserve energy in
response to the increased metabolic cost of sleep deprivation on
nightshift 1, as similar findings of reduced total daily EE are ob-
served during recovery sleep after total sleep deprivation (19).
Possible contributing mechanisms for decreased EE at the begin-
ning of the daytime sleep opportunity include reduced resting
metabolic rate (31) and sleep-induced decreases in the metabolic
hormones cortisol, norepinephrine, and epinephrine that typically
increase EE (32, 33) and are highest at this circadian time (5).
During sleep, circulating cortisol, norepinephrine, and epinephrine
levels are reduced and morning sleep after the nightshift could
decrease these hormones (34, 35), leading to decreased EE. Our
finding of shorter REM latencies during daytime sleep may be
associated with reduced catecholamine levels, thereby potentially
decreasing EE (34). These possible mechanisms, however, cannot
explain why EE is lower before daytime sleep. We did not measure
cortisol and catecholamines, and further studies are needed to
study these and other potential mechanisms that decrease EE
when sleep occurs at inappropriate circadian times.
Satiety and Appetitive Hormones. Appetitive hormones are altered
during circadian misalignment (reduced leptin) (5, 18) and in-
sufficient sleep (reduced leptin and increased ghrelin) (2, 36)
when subjects are given meals designed to meet energy balance
at baseline. Appetite increases have been reported in response to
insufficient sleep (2, 36). Our findings of decreased leptin and
PYY levels, with no change in ghrelin, would be hypothesized to
increase hunger ratings as seen with insufficient sleep (2, 36), yet
we observed decreased hunger ratings during the nightshift
McHill et al.
Day 3: nightshift 1 Day 4: nightshift 2 Day 5: nightshift 3
(n = 14) (n = 14) (n = 13)
212.1 (9.6) 168.2 (6.3)*, 200.1 (9.5)
91.0 (6.9) 99.3 (6.9){ 81.3 (6.8)
61.2 (4.4) 53.0 (3.3)*, 59.8 (3.6)
23.5 (9.7) 67.3 (9.8)*, 29.9 (11.1)
28.5 (5.1) 37.5 (5.5) 20.6 (6.0)
9.0 (4.9) 16.5 (3.6) 8.6 (2.0)
schedule. The decrease in leptin levels may be associated with
the negative fat balance observed. Also, percent change in leptin
was larger than the change reported after 3 d of 30% food re-
striction in healthy subjects (37), suggesting that the leptin
change we observed is physiologically meaningful. Our findings
suggest a disassociation between the peripheral appetitive hor-
mones examined and subjective hunger during acute circadian
misalignment. Why hunger was not increased may be related to
circadian mechanisms promoting satiety at night (38), lack of a
change in ghrelin, or changes in other peripheral satiety hormones
not measured such as cholecystokinin and insulin. Our findings
also provide further evidence that leptin and ghrelin are primarily
modulated by wakefulnesssleep, feedingfasting cycles compared
with circadian phase (5, 18). Further, our findings demonstrate
that the daily pattern in PYY levels is also primarily wakefulness
sleep versus circadian-driven.
How Circadian Misalignment May Contribute to Weight Gain. Knowl-
edge about potential mechanisms for increased risk of weight gain
and obesity in nightshift workers is important for developing evi-
dence-based treatment strategies. Self-reported total daily food
PHYSIOLOGY
intake is similar in day and shiftworkers (9). Thus, other mecha-
nisms may be needed to help explain weight gain in shiftworkers.
Our findings suggest that circadian misalignment during nightshift
schedules may disturb metabolic physiology and contribute to
adverse metabolic health outcomes by reducing total daily EE.
Specifically, the 217 to 247 kJ (5259 kcal) lower total daily EE
on nightshifts 2 and 3, if recurrent without a reduction in food
intake, would contribute to weight gain, because as little as 209
kJ (50 kcal) excess calorie storage per day can increase weight
over time (21). Further, if increased exhaustion and fatigue levels
(27) associated with shiftwork results in reduced physical activity
levels, this would promote positive energy balance and weight
gain. The decrease in TEF observed after a late meal applies to
shiftworkers and also to day workers who eat later at night and
have a higher risk of obesity (22). Further research is needed to
extend current findings to include all components of energy bal-
ance, in particular the effects of dietary choices and timing of
meals on risk of obesity in shiftworkers.
Methods
Subjects. Fourteen healthy subjects (eight women; aged 26.4 1.2 y, mean
SD, with BMI 22.7 0.5 kg/m2 and percent body fat 27.4 2.2% as de-
termined by dual-energy X-ray absorptiometry; Prodigy Advance; GE Lunar)
completed the protocol. Data from two additional subjects who did not
complete the protocol are not included, one due to claustrophobia in the
whole-room calorimeter and the other to gastrointestinal discomfort that
prevented meal consumption. Procedures were approved by the scientific and
advisory review committee of the Colorado Clinical and Translational Sciences
Institute, the Colorado Multiple Institutional Review Board (IRB), and the
University of Colorado Boulder IRB. Subjects gave written informed consent
PNAS | December 2, 2014 | vol. 111 | no. 48 | 17305
 psychiatric, or sleep disorder, current smoker, pregnancy, or a habitual sleep
A 1400
duration of <7 h or >9.25 h. Subjects were deemed healthy based on clinical
1200 history, physical examination, psychological tests, a psychological interview
with a trained clinician, blood chemistries (complete blood cell count and
1000 comprehensive metabolic panel), clinical electrocardiogram, and medication-
free status. No subjects reported regular night work in the preceding year or
Ghrelin (pg/ml)

800 crossing more than one time zone in the previous 3 wk. Subjects were mini-
mally physically active before the CTRC procedures to minimize the detraining
600 effects of the posture controlled study. Women were not scheduled to start
the study according to menstrual phase.
400
Study Protocol. For 1 wk before admission to the University of Colorado
200 Hospital (UCH) CTRC, subjects maintained a self-selected sleep schedule of 8 h
per night and caffeine, alcohol, nicotine, and over-the-counter medication
0 use was proscribed. The sleep schedule was verified via continuous wrist
actigraphy with light-exposure monitoring (Actiwatch-L; Mini-Mitter/
21 Respironics), sleepwakefulness logs, and call-in bed and waketimes to a
B time-stamped voice recorder. Drug use was determined by self-report and
18 verified by urine toxicology at screening and by urine toxicology and a
breath alcohol tester (Lifeloc Technologies; model FC10) upon CTRC admis-
15 sion. Three days before CTRC admission, exercise was proscribed and subjects
were provided a 3-d outpatient diet designed to meet their individual daily
Leptin (pg/ml)

12 caloric needs as determined from resting metabolic rate with a 1.5 activity
factor (9). Subjects were instructed to consume meals provided and nothing
9 else but water to ensure energy balance at the start of the CTRC protocol.
Subjects lived at the UCH CTRC for 6 d to simulate a daytime work
6 followed by a 3-d nightshift schedule (Fig. 1). A polysomnography (PSG)-
recorded 8-h nighttime sleep opportunity at habitual bedtime verified that
3 subjects were free from sleep disorders. Following the sleep disorders
screening night, subjects were awakened at habitual waketime and main-
0 tained a constant posture protocol with brief breaks: seated semirecumbent
posture in a hospital bed with the head raised to 35, room temperature
maintained at 2224 C, and dim lighting (<1 lx in the angle of gaze, <5 lx
C 140
max) during scheduled wakefulness and 0 lx during scheduled sleep. Day 2
120 served as a baseline day shift, with 16 h of daytime wakefulness and an 8-h
nighttime sleep opportunity. This procedure permitted subjects to sleep at
100 their habitual circadian phase at baseline. Day 3 served as the transition to
working nightshift 1. Subjects were awakened at habitual waketime and
PYY (pg/ml)

80 scheduled to a 2-h afternoon nap opportunity before nightshift 1. After

nightshift 1, subjects were provided an 8-h daytime sleep opportunity that
60 began 1 h after their habitual baseline waketime. This was followed by
nightshifts 2 and 3 (Fig. 1). After nightshift 3, subjects were provided a non
40 PSG-recorded 8-h sleep opportunity and discharged from the CTRC.
Subjects received scheduled meals (percent daily caloric intake: 30%
20 breakfast, 30% lunch, 30% dinner, 10% snack) at 1.5, 5.5, 10.5, and 14.5 h
postawakening on day 2 (relative clock hour 0930, 1330, 1830, and 2230
0 hours for a subject with scheduled waketime arbitrarily assigned a value of
0800 hours and all other times referenced to this value; actual clock hour
was determined by the participants habitual wakefulnesssleep schedule
D 49
assessed during ambulatory baseline) and on days 4 and 5 (relative clock
42 hour 1730, 2130, 0330, and 0730 hours). Meals were similar across days (e.g.,
the food served for breakfast was the same each day) and subjects were
35 instructed to consume all food provided. Because day 3 simulated the
Hunger Ratings (mm)

transition day to nightshift 1 with a 2-h afternoon nap opportunity, the

28
21
14
7
0 indirect calorimetry during days 25 (20, 39). Subjects carbon dioxide (CO2)
Baseline Nightshift 1
Transition
Fig. 4. Appetitive hormones (AC) [n = 14; P values, planned comparisons
for day (24 h), one-tailed] and hunger ratings (D) (n = 14; P values, planned
comparisons for day, two-tailed). Solid lines represent significant differences
at the end of each line (modified Bonferroni, P < 0.025), and the dashed line
represents a nonsignificant trend (P = 0.03). Error bars are SEM.
and then underwent health screening at the Sleep and Chronobiology Labo-
ratory and the University Colorado Boulder Clinical Translational Research Cen-
ter (CTRC). Exclusion criteria consisted of diagnoses with any known medical,
timing and percent of daily caloric intake (30% breakfast, 25% lunch, 10%
snack, 25% dinner, 10% snack) were spread across the day; these meals were
given at 1.5, 5.5, 10.5, 15.5, and 20.5 hours (relative clock hour 0930, 1330,
1830, 2230, and 2330 hours) after scheduled awakening. Dinner was a cold
meal kept in a refrigerator next to the bed so subjects did not change
posture, permitting TEF analysis.
Measures. EE and macronutrient utilization were determined via whole-room
Nightshift 2 Nightshift 3 production and oxygen (O2) consumption were assessed to calculate EE and
respiratory quotient (RQ) by measuring gas concentration differences entering
and exiting the calorimeter with fuel cell-based dual-channel O2 analyzers (FC-2
Oxzilla; Sable Systems International) and two infrared CO2 analyzers (CA-10
CO2 analyzers; Sable Systems International) (39). Monthly propane combustion
tests validated the precision and accuracy of the gas concentration measure-
ments with average recoveries of 99%. Nonprotein carbohydrate and fat
utilization were determined from O2 consumption and RQ (40), and protein
utilization was calculated using 24-h urine total nitrogen (41).
Leptin, ghrelin, and PYY levels were assessed from blood samples drawn
every 2 h on days 25. Melatonin was assessed every 2 h during the daytime
and hourly at night starting at 13 h after habitual waketime (relative clock

17306 | www.pnas.org/cgi/doi/10.1073/pnas.1412021111 McHill et al.

hour 1900 hours) as a marker of internal circadian time. Blood was drawn
using a 12-foot extension tubing connected to an indwelling venous cath-
eter and kept patent with a heparinized saline drip (20). Extension tubing
through a porthole in the calorimeter allowed for blood sampling without
entering the room or disrupting scheduled sleep. Hunger ratings were de-
termined via visual analog scales collected every 2 h starting at 2 h post-
waketime and ending 2 h before sleep.
Sleep and wakefulness recordings, including sleep disorders screen night 1,
were obtained using Siesta digital recorders (Compumedics). Sleep latencies
were defined as follows: SOL, time from lights out to the onset of three
continuous epochs of PSG-defined sleep; LPS, time from lights out to the
onset of 10 continuous minutes of PSG-defined sleep; latency to SWS (SWSL),
time from LPS to SWS; and REML, time from LPS to REM sleep.
Data Analysis. EE and macronutrient utilization were averaged hourly during
scheduled wakefulness and sleep and averaged for the 24-h day. Total daily
carbohydrate balance was calculated accounting for fiber intake. EE was also
calculated for consolidated sleep and wakefulness stages during scheduled
sleep opportunities as in ref. 19 and for all scored sleep epochs. Specifically,
sleep and wakefulness stages (WPSO, WASO, stage 1 sleep, stage 2 sleep,
SWS, and REM) were matched with EE values that were offset by 2 min to
account for the lag-time response of the whole-room calorimeter relative to
the timing of the sleep recording (19, 42). Also see Table S2. Blood data were
aligned and plotted beginning from scheduled wakefulness. TEF was mea-
sured as a deviation from the EE 1 h before dinner (premeal baseline, sub-
jects seated for 4 h before baseline measurement) for the 3.5-h postdinner
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McHill et al.
consumption and analyzed as the percentage of the caloric content of the
dinner. Appetitive hormones were analyzed for 24-h and 2-h averages.
Protein utilization was unavailable for one subject on nightshift 3 and thus
excluded from the macronutrient utilization analysis for that day. Three
subjects on nightshift 2 and two subjects on nightshift 3 did not have
documented completion times of dinner and were therefore excluded from
the TEF analysis for those days. The sleep recording for one subject was
missing for the sleep opportunity before nightshift 2 and therefore was
excluded from the sleep analyses for that day.
Data were analyzed via mixed-model ANOVAs with day, relative clock
time, and/or sleep stage as fixed factors and subject as a random factor using
STATISTICA version 10.0 (StatSoft). One-tailed a priori directional planned
comparison dependent t tests, with modified Bonferroni correction for mul-
tiple planned comparisons to reduce type 1 error (43), examined the hy-
pothesized reduction in fat utilization, increase in sleep opportunity EE, and
changes in 24-h appetitive hormones. Two-tailed planned comparisons for
other measures were also computed. Exploratory post hoc analyses were
performed for fat utilization and sleep opportunity EE after the failure to
confirm hypotheses. Data in the text are presented as mean SEM.
ACKNOWLEDGMENTS. We thank the participants, the University Colorado
Boulder Clinical Translational Research Center staff, and B. Birks, B. Smith,
B. Brainard, B. Griffin, T. Dear, and G. Wright, for study assistance. This research
was supported by NIH R21 DK092624, NIH 1UL1 RR025780, P30 DK048520,
and the Undergraduate Research Opportunities Program in collaboration
with the Howard Hughes Medical Institute and Biological Sciences Initiative.
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PNAS | December 2, 2014 | vol. 111 | no. 48 | 17307
Supporting Information
McHill et al. 10.1073/pnas.1412021111
SI Methods
Subjects. In addition to subject inclusion information presented in
the main text, young ovulating women with a history of regular
menstrual cycles and no history of prior gynecological pathology
were selected. Half of the women used oral contraceptives, and
those not currently using oral contraceptives were required to be
free from oral contraceptive use for more than 3 mo before
participation and to not have irregular cycle lengths. Subjects with
mood disorders, including late luteal phase dysphoric disorder
(determined at clinical interview), were excluded. Menstrual
phase logs indicated that women started the study in the week of
menses or on the first day of the follicular phase (n = 3) or during
the luteal phase (n = 5). For those women taking oral contra-
ceptives, the study was started in the pseudo follicular phase
(n = 3) and luteal phase (n = 1). Although we included near-
equal numbers of men and women in our study, that we did not
control for or determine the influence of menstrual cycle phase
or oral contraceptives on our outcome measures is a limitation.
Chronotype was not an inclusion/exclusion criterion. Chronotype
estimates derived from the Morningness-Eveningness Question-
naire (1) were as follows: One was a definite morning type, four
were moderate morning types, seven were intermediate types, and
two were moderate evening types.
Meals. Diets were prepared by the CTRC Nutrition Core and
contained macronutrient contents recommended by the Ameri-
can Heart Association (30% fat, 55% carbohydrate, and 15%
protein) and no caffeine. Inpatient meals were the same each day
for each individual subject and typically consisted of some vari-
ation of the following: breakfast contained eggs, toast, and juice;
lunch contained a turkey sandwich, potato chips, and fruit; dinner
contained a salad with meat and a roll; and the snack contained
a cracker with cheese. Outpatient meals differed from inpatient
meals and between subjects. Change in subjects preferences for
food provided across the study was not assessed. CTRC die-
1. Horne JA, Ostberg O (1976) A self-assessment questionnaire to determine morning-
ness-eveningness in human circadian rhythms. Int J Chronobiol 4(2):97110.
2. Weiss R, Fong AK, Kretsch MJ (2003) Adapting ProNutra to interactively track food
weights from an electronic scale using ProNESSy. J Food Compos Anal 16(3):305311.
3. Rechtschaffen A, Kales A (1968) A Manual of Standardized Terminology, Techniques
and Scoring System for Sleep Stages of Human Subjects (Brain Research Institute,
University of California, Los Angeles).
4. Spiegel K, Tasali E, Penev P, Van Cauter E (2004) Brief communication: Sleep curtail-
ment in healthy young men is associated with decreased leptin levels, elevated ghrelin
levels, and increased hunger and appetite. Ann Intern Med 141(11):846850.
5. Taheri S, Lin L, Austin D, Young T, Mignot E (2004) Short sleep duration is associated with
reduced leptin, elevated ghrelin, and increased body mass index. PLoS Med 1(3):e62.
McHill et al. www.pnas.org/cgi/content/short/1412021111
ticians determined the caloric and macronutrient content of
meals using ProNutra software (2).
Measures. EE is provided in units of kcal (kilocalorie) and kJ
(kilojoule) per minute; 1 kcal = 4.184 kJ. Sleep recordings were
obtained from C3-A2, C4-A1, O1-A2, O2-A1, F3-A2, F4-A1,
right and left electrooculogram (EOG), chin electromyogram
(EMG), and electrocardiogram (ECG). The sleep disorders
screen tested for sleep apnea, periodic limb movements, noctur-
nal epilepsy, and other sleep-related movement disorders based
on standardized EEG, airflow, respiratory effort, and leg EMG
measurements. Sleep was manually scored in 30-s epochs ac-
cording to standard guidelines from brain region C3-A2 (3) or C4-
A1 if C3-A2 contained an artifact. Approximately 515 mL of
blood was drawn throughout the duration of the protocol, in-
cluding blood taken at screening. Blood samples were immedi-
ately centrifuged (2,0003,000 g) and subsequently stored at
80 C until assayed. Serum radioimmunoassays (RIAs) for
ghrelin sensitivity 93 pg/mL, within-assay coefficient of variation
(CV) 4.5%; peptide-YY sensitivity 10 pg/mL, within-assay CV
5.3%; and leptin sensitivity 0.5 ng/mL, within-assay CV 5.9%
(Millipore) were performed by the CTRC core laboratory; mel-
atonin sensitivity 2.3 pg/mL, within-assay CV 13.4% (LDN Mel-
atonin Direct RIA; Rocky Mountain Diagnostics) was performed
by the Sleep and Chronobiology Laboratory. We examined total,
not acetylated, ghrelin, because total ghrelin levels have been
reported to respond to sleep loss and may contribute to increased
hunger (4, 5). Assessment of acetylated ghrelin and of various
acylghrelin isoforms should be considered in future studies, as
they are more physiologically relevant in regard to ghrelin re-
ceptor activation and effects on food consumption, weight gain,
and energy homeostasis compared with total ghrelin levels (6, 7).
Furthermore, changes in other metabolic hormones not exam-
ined (e.g., glucagon-like-peptide, cholecystokinin, cortisol, tes-
tosterone, and catecholamines) (8, 9) could have contributed to
the current findings.
6. Heppner KM, et al. (2012) Acylation type determines ghrelins effects on energy ho-
meostasis in rodents. Endocrinology 153(10):46874695.
7. Spiegel K, Tasali E, Leproult R, Scherberg N, Van Cauter E (2011) Twenty-four-hour
profiles of acylated and total ghrelin: Relationship with glucose levels and impact of
time of day and sleep. J Clin Endocrinol Metab 96(2):486493.
8. Spiegel K, Leproult R, Van Cauter E (1999) Impact of sleep debt on metabolic and
endocrine function. Lancet 354(9188):14351439.
9. Leproult R, Van Cauter E (2011) Effect of 1 week of sleep restriction on testosterone
levels in young healthy men. JAMA 305(21):21732174.
1 of 4
 Day 2- Baseline
Day 3- Nightshift 1
Day 4- Nightshift 2
100 Day 5- Nightshift 3

80

Melatonin (pg/ml)
60

40

20

0 Nap Nightshift wakefulness

Daytime sleep Habitual Sleep

08 16 24 08

Relative Clock Hour

Fig. S1. Hourly melatonin levels (n = 14). The gray line represents day 2 (baseline), the black line is day 3 (transition to nightshift 1), the blue line is day 4
(nightshift 2), and the red line is day 5 (nightshift 3). Time of day is plotted as relative clock hour with scheduled waketime arbitrarily assigned a value of 0800
hours. Black bars represent scheduled sleep, and the yellow bar represents scheduled nighttime wakefulness during shiftwork. Error bars are SEM. There was
a nonsignificant change in dim-light melatonin onset between days (P > 0.08).

Day 2- Baseline
Day 4- Daytime sleep
Prior to Nightshift 2
7 Day 5- Daytime sleep 1.7
Energy Expenditure (kjoules/min)

Energy Expenditure (kcal/min)

Prior to Nightshift 3
*
6 * * 1.4
* * *
* *
5 * 1.2

4 1.0

3 0.7

2 0.5

1 0.2

0 0
WPSO WASO Stage 1 Stage 2 SWS REM
Sleep Stage
Fig. S2. Energy expenditure per sleep stage (n = 14; P values, planned comparisons for day, two-tailed). Solid lines and asterisks represent significant (P < 0.05)
differences between stages at the end of each line. Error bars are SEM.

McHill et al. www.pnas.org/cgi/content/short/1412021111 2 of 4

Fig. S3. Average 2-h appetitive hormones (AC) (n = 14; two-tailed). Planned comparisons were calculated by dependent t test (P < 0.05). *Differences
between baseline and the transition to nightshift 1; #differences between baseline and nightshift 2; differences between baseline and nightshift 3; dif-
ferences between the transition to nightshift 1 and nightshift 2; differences between the transition to nightshift 1 and nightshift 3; $differences between
nightshift 2 and nightshift 3. Black arrows indicate the timing of meals. B, breakfast; D, dinner; L , lunch; S, snack; italicized D and S indicate different meals
given at that time on transition to the first nightshift. Black bars represent sleep opportunities. Note that the nap only occurs on the transition to the first
nightshift; the open bar represents sleep deprivation. Error bars are SEM. Hourly ghrelin levels fluctuated across the day. Average 2-h leptin levels were
generally lower during wakefulness than during sleep. Regardless of whether sleep occurred at night or during the daytime, average 2-h PYY levels were
higher during wakefulness than during sleep.

McHill et al. www.pnas.org/cgi/content/short/1412021111 3 of 4

 Table S1. Sleep architecture for nighttime baseline and daytime shiftwork sleep opportunities
Baseline nighttime sleep Daytime sleep before Daytime sleep before
Measure opportunity (n = 14) nightshift 2 (n = 13) nightshift 3 (n = 14)

Percent of recording time

Stage 1 4.1 (0.5) 3.6 (0.4) 4.0 (0.6)
Stage 2 55.6 (1.9) 46.8 (2.2)* 47.1 (2.5)
SWS 12.5 (1.5) 14.3 (1.9) 14.1 (1.7)
REM 18.3 (1.3) 14.3 (1.3)* 16.1 (1.0)
SE 90.4 (2.0) 79.0 (2.4)* 81.3 (2.2)
Minutes of recording time
Stage 1 19.6 (2.6) 17.2 (1.9) 19.2 (2.7)
Stage 2 266.6 (9.3) 224.7 (10.8)* 226.0 (12.1)
SWS 61.4 (7.1) 70.1 (8.7) 67.4 (8.0)
REM 87.9 (6.3) 68.6 (6.3)* 77.1 (4.8)
WASO 34.8 (9.4) 95.6 (11.5)* 85.2 (10.1)
TST 433.9 (9.7) 379.0 (11.7)* 389.6 (10.8)
SOL 9.9 (1.2) 5.6 (0.9)* 4.7 (1.1)
LPS 13.3 (1.7) 6.0 (1.0)* 4.9 (1.3)
SWSL 17.9 (2.5) 20.3 (3.6) 16.9 (1.9)
REML 83.4 (8.0) 52.1 (4.7)* 54.6 (1.4)
Duration of awakenings 1.5 (0.3) 5.2 (1.1)* 4.9 (1.0)
No. of awakenings 22.3 (2.7) 21.6 (3.0) 23.4 (3.0)

Findings for changes in both daytime sleep opportunities are described in the main text. Daytime sleep also displayed a nonsignif-
icant trend for increases in both percent and minutes of SWS (P = 0.08). Values in parentheses are SEM. LPS, latency to persistent sleep;
REM, rapid eye movement; REML, latency to REM sleep; SE, sleep efficiency; SOL, sleep-onset latency; SWS, slow-wave sleep; SWSL,
latency to SWS; TST, total sleep time; WASO, wakefulness after sleep onset.
*Denotes P < 0.05 between baseline night sleep opportunity and sleep before nightshift 2.

Denotes P < 0.05 between baseline night sleep opportunity and sleep before nightshift 3.

Table S2. ANOVA F values for energy expenditure comparisons between sleep stages for
individual sleep opportunities
Measure Stage 1 Stage 2 SWS REM WPSO

Baseline nighttime sleep

opportunity (n = 14)
Stage 2 0.75
SWS 0.01 0.72
REM 0.89 0.09 0.53
WPSO 9.80* 12.33* 19.90* 12.99*
WASO 3.34 11.64* 4.18 8.68* 4.39
Daytime sleep before
nightshift 2 (n = 13)
Stage 2 1.87
SWS 3.34 0.82
REM 0.02 1.18 3.18
WPSO 16.76* 6.99* 6.37* 8.89*
WASO 16.84* 25.35* 25.62* 9.20* 27.91*
Daytime sleep before
nightshift 3 (n = 14)
Stage 2 1.78
SWS 0.06 1.04
REM 1.97 0.26 0.96
WPSO 11.09* 5.28* 6.92* 7.17*
WASO 4.20 28.49* 12.08* 38.78* 20.81*

Energy expenditure corresponding to consolidated stage 2 and slow-wave sleep epochs were binned and
averaged for 15 min of continuous sleep for that stage, with the exception of four subjects who did not have
15 min of continuous SWS and therefore the maximum time for each subject was used (the minimum bin size
was 4 min for one subject); EE bins were 9.5 min for rapid eye movement, 1 min for stage 1, and 30 s for
wakefulness prior to sleep onset and wakefulness after sleep onset.
*Denotes P < 0.05 between stages.

Denotes 0.07 < P < 0.1 between sleep stages.

McHill et al. www.pnas.org/cgi/content/short/1412021111 4 of 4