DOT/FAA/AM-02/20

Albert Boquet

Crystal E. Cruz

Thomas E. Nesthus

Cristy A. Detwiler

William R. Knecht

Kali A. Holcomb

Civil Aerospace Medical Institute

Federal Aviation Administration

Oklahoma City, OK 73125

November 2002

Final Report

This document is available to the public

through the National Technical Information

Service, Springfield, VA 22161.

A Laboratory Comparison of

Clockwise and Counter-Clockwise

Rapidly Rotating Shift Schedules,

Part III: Effects on Core Body

Temperature and Neuroendocrine

Measures

Office of Aerospace Medicine

Washington, DC 20591
NOTICE

This document is disseminated under the sponsorship of

the U.S. Department of Transportation in the interest of

information exchange. The United States Government

assumes no liability for the contents thereof.
i

Technical Report Documentation Page

1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-02/20

4. Title and Subtitle 5. Report Date

A Laboratory Comparison of Clockwise and Counter-Clockwise Rapidly November 2002

Rotating Shift Schedules, Part III: Effects on Core Body Temperature and

Neuroendocrine Measures 6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No.

Boquet A, Cruz CE, Nesthus TE, Detwiler CA, Knecht WR, and Holcomb

KA

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS)

FAA Civil Aerospace Medical Institute

P.O. Box 25082

Oklahoma City, OK 73125 11. Contract or Grant No.

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered

Office of Aerospace Medicine

Federal Aviation Administration

800 Independence Ave., S.W.

Washington, DC 20591 14. Sponsoring Agency Code

15. Supplemental Notes

This report was performed under Task HRR518.

16. Abstract

Most researchers suggest that shift rotation in a forward or clockwise direction produces less disruption of

circadian rhythms than those that rotate in a backward or counter-clockwise direction. This is based upon

extrapolation from quasi-experimental studies of shift-workers and research on the effects of jet lag, which

indicate that westward travel results in less disruption of circadian rhythms. The effect of direction of rotation

on cortisol, melatonin, and core body temperature was examined in participants randomly assigned to either a

clockwise or counter-clockwise shift rotation. Twenty-eight participants worked a day shift (0800-1600) for

one week followed by either a clockwise (n=14) or counter-clockwise (n=14) shift rotation for two weeks.

Participants wore a flexible rectal temperature sensor for the three weeks of the study and were allowed to

remove the sensor for 90 minutes each day. Saliva samples were collected at the end of the baseline week for

later assay for melatonin and cortisol, and were time-locked to collection times during the two “shiftwork”

weeks. No group differences were found for cortisol for either of the workweeks. The clockwise group,

however, had a significantly greater increase in melatonin during the early morning shift, compared with the

counter-clockwise group. Finally, the analyses of core body temperature revealed a significantly lower

amplitude and a delay of the acrophase for the counter-clockwise group during both weeks of testing. While

cortisol levels did not appear to differ based on direction of shift rotation, there were inherent differences with

melatonin secretion and core body temperature. Precisely why such differences exist remains unclear but may

be similar to that seen with circadian resynchronization during westward travel. Furthermore, it is not clear if

these differences would persist in individuals exposed to longer periods of shift rotations.

17. Key Words 18. Distribution Statement

Document is available to the public through the

National Technical Information Service;

Shiftwork, Cortisol, Melatonin, Core Body Temperature,

Circadian Rhythms, Clockwise, Counter-Clockwise

Springfield, VA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price

Unclassified Unclassified 19

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

iii

ACKNOWLEDGMENTS

The authors gratefully acknowledge Pamela Della Rocco, Ph.D. for her foundational work in

shiftwork and fatigue research at the Civil Aerospace Medical Institute, for proposing this project as part

of the larger research program, and for her contributions to the protocol for this study. In addition, we

thank Mr. Blake Sanders for his contribution to the data collection and test administration for this study,

Ms. Susan Buriak for her effort in collecting articles relevant to melatonin and cortisol, and Mrs. Jane

Brooks for providing the food service for the study. We also thank Dr. LeRoy Blank and his students,

Ms. Karen Bailey and Ms. Whitney Smith, from the University of Oklahoma Chemistry Department,

for providing the melatonin and cortisol assays for the study.

1

A LABORATORY COMPARISON OF CLOCKWISE AND COUNTER-CLOCKWISE

RAPIDLY ROTATING SHIFT SCHEDULES, PART III:

EFFECTS ON CORE BODY TEMPERATURE AND NEUROENDOCRINE MEASURES

Because of the increasing industrialization of our

society, shiftwork is necessary in many occupations.

Accordingly, much research has been conducted on

the effects of shiftwork as it relates to the health and

well being of individuals engaged in such activities.

The underlying rationale is that shiftwork presents a

potential source of strain that may ultimately take a

toll on the shiftworker, in any one of a number of

ways. For instance, Monk (1988) has postulated a

model of shiftwork strain composed of three interactive components: sleep, issues associated with the

biological clock, and social/domestic factors. All three

components play an interactive role in determining

how someone will cope with the demands of shiftwork.

Given the complexity of managing shiftwork and

performance in the workforce, the role of management and industry is to try to implement work schedules that will result in the best compromise between

productivity and employee satisfaction, health, and

performance. Debate in the literature, bolstered in

part by a large body of research on jet-lag, has suggested that shift schedules that change in the direction

of the circadian cycle (i.e., clockwise or forward rotations), result in less physiological and mental disruption than those that rotate in the opposite direction

(Monk, 1986; Barton & Folkard, 1993). If this is true,

perhaps these same kinds of schedules might result in

less of a physical challenge, thus ameliorating the

social and emotional impact of working different

shifts. Nevertheless, while jet-lag studies have shown

that individuals adapt more quickly to westward travel

(Wegman & Klein, 1985), jet travel and shiftwork are

only partially analogous. That is, exposure to daylight

acts as an exogenous cue thereby promoting adaptation to new time zones as one travels. However, these

same daylight cues are often in conflict with shiftwork

schedules. Consequently, we are left with little empirical evidence to support the claim that clockwise

rotations result in less disruption and therefore, less

physical challenge to the shiftworker (Barton &

Folkard, 1993).

Addressing the importance of maintaining circadian rhythmicity by shift schedule design, Lavie,

Tzischinsky, Epstein, and Zomer, (1992) compared

workers on clockwise and counter-clockwise, 5-dayper-shift schedules. They reported better synchronization of sleep-wake cycles and earlier sleep times

following midnight shifts for those individuals in the

clockwise rotation. In addition, they infer that the

quicker adaptation of biological rhythms of the clockwise group may be important in enhancing health

outcomes in this group. However, the lack of statistically significant results implies that the authors may

have overstated their findings.

In addition, there is evidence that people never

fully adapt to shifts other than standard daytime

hours (Naitoh, Kelly, & Englund, 1990). Even those

individuals on permanent night shifts show poor

adjustment in circadian markers and overall sleep

quality compared with their day-working cohorts.

The reasons for this include, but may not be limited

to, reverting to a “normal” schedule on their days off,

difficulty sleeping in the daytime, individual differences in ability to adapt to shiftwork, and the mismatch of schedule with exogenous cues such as

daylight.

While individuals may never fully adapt to shiftwork

physiologically, there is some evidence that at least

some individuals tolerate shiftwork better than others

(Costa, Lievore, Casaletti, & Gaffuri, 1989). In a

study of 24 participants matched for age and work

experience, Costa et al. found that those individuals

who demonstrated greater flexibility in sleep habits,

resistance to drowsiness, and reported lower manifest

anxiety had a greater tolerance to shiftwork. Notably,

however, these characteristics did not affect the adjustment of oral temperature to night shiftwork, indicating that physiological adaptation likely did not

take place.

Perhaps oral temperature, as used in the Costa et al.

study isn’t the best measure of physiological adaptation to shiftwork. One alternative is to use cortisol as

a physiological marker. Cortisol is the principal

gluccocorticoid of the adrenocortical hormones with

widespread systemic effects. The most notable of

these is its ability to markedly stimulate gluccogenesis;

hence, its classification as a gluccocorticoid. Cortisol

has a relatively static diurnal rhythm, which according to
2

Munck, Guyre, and Holbrook (1984), has both diurnal and metabolic functions, is controlled by hypothalamic signals, and is regulated via a sensitive negative

feedback loop.

What makes cortisol a particularly good marker in

studies of circadian disruption is its release during

emotional stress. For example, shiftwork tolerance

was investigated by Hennig, Kieferdorf, Moritz, Huwe,

and Netter (1998) in a sample of 24 night shiftworkers.

They found a reversal of the circadian pattern for

cortisol in 18 of the 24 workers after the fifth night of

shiftwork. The 6 non-adapters demonstrated less sleep,

greater variability in their sleep patterns, and higher

neuroticism scores. The authors concluded that there

are individual differences in shiftwork tolerance as

revealed by cortisol secretion.

In a similar study, Hale, Williams, Smith, and

Melton (1971) found adrenocortical activity late in

the morning shift among a sample of air traffic controllers at O’Hare Airport in Chicago. However, following the night shift, a depression in adrenocortical

activity was observed. As a result, the authors suggested that nocturnal wakefulness acted as a stressor,

accounting for some of the elevation noted in both

control and experimental groups.

The relationship between cortisol levels and

shiftwork is particularly apparent in cortisol responses

to sleep deprivation, an inherent characteristic of

working night shifts. Leproult, Copinschi, Buxton,

and Van Cauter (1997) examined male participants in

normal sleep schedules, partial sleep deprivation, and

total sleep deprivation conditions. Both partial and

total sleep deprivation resulted in elevated cortisol

levels on the second recovery day following deprivation. This was accompanied by a delay in the recovery

of the lull in cortisol secretion, suggesting a disruption of the negative feedback system of the hypothalamic-pituitary-adrenocortial (HPA) axis.

Another commonly used circadian marker is body

temperature because of its highly entrained rhythm,

regulated by sensitive negative feedback loops in temperature-regulating centers of the hypothalamus

(Guyton & Hall, 2000). This highly efficient system

has proven a useful circadian marker in studies like

that of Reinberg, Vieux, Ghata, Chaumont, and

Laporte (1978), who found that low amplitude in the

oral temperature rhythm may reflect the ability to

adjust to shiftwork. In contrast, Knauth and Harma

(1992) found that large amplitude in oral temperature

rhythm prior to shiftwork was positively associated

with health-related indices of shiftwork tolerance.

Like body temperature, melatonin secretion has

proven to be a reliable measure of intrinsic circadian

rhythmicity. However, unlike body temperature,

melatonin is relatively insensitive to exogenous cues,

other than light. As such, it may be a more reliable

marker of circadian rhythmicity than body temperature. Produced by the pineal gland following its synthesis from tryptophan, concentrations of melatonin

are relatively high at night and at their lowest levels

during the day with the rhythm entrained by photic

stimuli, exercise, certain drugs, and exogenous melatonin (Guyton & Hall, 2000).

Roden, Koller, Pirich, Vierhapper, and Waldhauser

(1993) examined the circadian melatonin profile for

permanent night shiftworkers, compared with matched

controls. The night shiftworkers as a group displayed

no adaptation of circadian profiles for either melatonin or cortisol. All workers scored high in job satisfaction, indicating that high shiftwork tolerance is

explained by more than circadian orientation.

The findings of Roden et al. (1993) are opposed to

others who found wide variations in melatonin profiles, particularly the onset of melatonin release, among

permanent night shiftworkers when compared to day

workers (Weibel, Spiegel, Gronfier, Follenius, &

Branden-Berger, 1997). Others have found forward

shifts in the mean peak of melatonin as well as a lack

of an identifiable acrophase during night work among

permanent night shiftworkers (Quera-Salva, Defrance,

Claustrat, DeLattre, & Guilleminault, 1996).

Melatonin has been shown to phase shift following

time zone changes, with the rate of return to its

baseline rhythm determined by how quickly the time

zones change (Harma, Laitinen, Partinen, & Suvanto,

1993). Following a round trip flight from Helsinki to

Los Angeles, the phase delay for melatonin peaked at

5 hours, 59 minutes. Resynchronization of the rhythm

had occurred within five days of the flight home,

leading the authors to recommend a five-day recovery

following numerous, rapid time zone transitions.

Reentrainment of the melatonin rhythm following

time zone transitions is dependent on cues in the new

time zone and direction of travel. Westward travel

results in faster resynchronization of the rhythm as the

system entrains to a period longer than a day. This is

analogous to a clockwise rotation, which ultimately

results in a longer day as the work week progresses.

Roach, Fletcher, Rodgers, and Dawson, (2000), followed 14 members of a surveillance patrol on a 13-day

routine flight. Their findings indicated that the onset

of melatonin phase was delayed in westward flight
3

with a slower recovery. Eastward flight resulted in a

phase advance of melatonin onset with faster subsequent recovery following small acute time zone

changes. While these findings are contrary to studies

showing faster recovery times and phase advances

following westward flight (Graeber, 1988), only melatonin onset was measured, thus the overall fit of the

curve may have been quite different.

In a study of the adaptation of permanent night

shiftworkers, Weibel et al. (1997) measured melatonin and core body temperature. They found that

shifting participants to a daytime sleep period resulted

in an uncoupling of the melatonin and temperature

rhythms, with temperature displaying a homogenous

pattern consistent with their usual sleep-wake cycle.

Of interest was the observation that adaptation to

night shiftwork was highly variable with respect to

melatonin. Thus, the two systems are differentially

affected by consistent nighttime work.

In a study of the relationship of gender to the

adjustment of circadian markers to night shiftwork,

Hakola, Harma, and Laitinen (1996) found no changes

in salivary melatonin levels following a rotation to

consecutive night shifts. While other markers such as

cortisol and body temperature exhibited an effect for

shift, no differences were noted for gender. Furthermore, no differences due to shift or gender were noted

for melatonin.

While subjective and behavioral indices of shiftwork

adaptation are important, they lack the rigor necessary

to objectively evaluate changes or shifts in the circadian rhythm of the individual. Biochemical markers

with relatively entrained circadian rhythms such as

cortisol and melatonin may provide useful and less

subjective measures of changes in diurnal patterns.

Temperature has also been used because of its sensitivity to changes in schedule. With this in mind, the

current investigation used core body temperature

(CBT) to test the effects of a clockwise vs. counterclockwise shift system on the circadian profile of 28

individuals in a simulated work environment. Salivary cortisol and melatonin were measured during

work to assess differences in production due to the

different work schedules. According to Aston-Jones,

Chen, Zhu, and Oshinsky (2001), CBT, melatonin, and cortisol may share a common biological

rhythm generator (the suprachiasmatic nucleas of

the hypothalamus).

METHODOLOGY

A 3-week protocol was designed for a laboratory

comparison of rapidly rotating clockwise and counterclockwise shifts, where schedule rotation represented

the between-groups factor in a mixed-model, repeated

measures design. Participants were run in groups of

five, with the direction of rotation balanced so the

first group was assigned to the clockwise rotation, the

second group was assigned to the counter-clockwise

rotation, etc. Although the protocol for the experiment was extensive including multiple computer tests,

physiological measures, and subjective measures, this

paper will focus on the temperature and biochemical

measures; therefore, only the procedures related to data

collection for these measures will be discussed in detail.

Participants

Demographics and Group Assignment

Thirty people between the ages of 20 and 55 (M = 41.2

years) were recruited and screened from the general

population to participate in the study. Participants signed

the informed consent form approved by the FAA Institutional Review Board and were paid for their participation in the study. Two participants withdrew from the

study before completing the protocol. Participants were

assigned to either the clockwise rotation condition (n =

14) or the counter-clockwise rotation condition (n =

14). Group assignments were made in the order that

participants were recruited and on the basis of their

availability. Participants in the clockwise rotation

included 7 males and 7 females, with an average age of

40.6 years (SD = 9.4 yrs.). Participants in the counterclockwise rotation included 5 males and 9 females

with an average age of 41.9 years (SD = 9.0 yrs.). All

participants were non-smokers and light- or nonusers of caffeine and alcohol. Additional details regarding the participant sample and their recruitment

and selection are reported elsewhere (Cruz, Detwiler,

Nesthus, & Boquet, 2002).

Procedures and Apparatus

Participants in the study reported to the laboratory

at the Civil Aerospace Medical Institute (CAMI) for

8 hours per day, 5 days per week, for 3 weeks. The first

week (Monday-Friday) for both rotation conditions

was comprised of day shifts (0800-1600). During this

week, participants were trained on computerized tasks

and habituated to the laboratory environment and to

wearing a physiological monitor. During the next 2
4

weeks, participants worked one of the shift rotation

schedules shown in Table 1, as determined by their

group assignment. Note that the clockwise rotation

allowed for 24 hours off at each shift rotation and a

48-hour weekend before returning to work again. By

comparison, the counter-clockwise rotation allowed

only 8 hours off at each shift rotation and an 80-hour

weekend before returning to work again.

On the first day of the study, participants were

oriented to the laboratory and a detailed daily schedule for the study. Two one-time questionnaires were

completed, a Morningness-Eveningness Questionnaire

(Horne & Ostberg, 1976) and a biographical questionnaire. In addition, participants were given physiological monitoring devices and daily logbooks, and

were trained on their use. Finally, participants were

trained on the Multiple Task Performance Battery

(MTPB) and the Bakan Vigilance Task. The physiological monitors and all sensors except the chest

band were worn 22.5 hours per day to accommodate

a 1.5-hour break for showers and leisure activities.

The only restriction was that the monitor should not

be removed while sleeping or napping. The chest band

sensor was only worn while working at the laboratory.

The Bakan Vigilance Task was administered at the

beginning and end of each workday, and the MTPB

was performed 3 times each day. The daily protocol is

presented in Table 2.

Table 2

Daily Experimental Protocol

Time (in hours) Activity

Start End

00:00 00:30 Download & initialize Miniloggers; Subjective ratings; Collect saliva

00:30 01:00 Bakan Session 1

01:00 02:30 MTPB Session 1

02:30 02:45 Subjective ratings; Collect saliva

02:45 03:15 Break

03:15 04:45 MTPB Session 2

04:45 05:00 Subjective ratings; Collect saliva

05:00 05:30 Meal Break

05:30 07:00 MTPB Session 3

07:00 07:30 Bakan Session 2

07:30 08:00 Download & initialize Miniloggers; Subjective ratings; Collect saliva

Table 1

Clockwise and Counter-Clockwise Shift Rotation Schedules

Clockwise Rotation Counter-Clockwise Rotation

Day Work Hours Hours

Between

Day Work Hours Hours

Between

Monday 0600-1400 16 Monday 1400-2200 16

Tuesday 0600-1400 24 Tuesday 1400-2200 8

Wednesday 1400-2200 16 Wednesday 0600-1400 16

Thursday 1400-2200 24 Thursday 0600-1400 8

Friday-Sat 2200-0600 48 Thur-Friday 2200-0600 80
5

Participants were instructed to treat their participation in the study as a full-time job, to refrain from

drinking alcohol or taking any drugs or medications

during the course of the study with the exception of

ibuprofen, birth control pills, estrogen replacement,

and non-drowsy formula allergy medications such as

ClaritinTM. In addition, participants were instructed

not to consume any caffeinated beverages or chocolate

and were not allowed to eat bananas during the study

because of the potential interference with the radioimmunoassays for cortisol. Diet was not otherwise

controlled in the study.

Participants were tested with the Intoxilyzer 9000TM

breath alcohol test at the beginning of each workday

to ensure compliance with the study protocol. None

of the participants tested positive during the study. A

final day of testing on Day 22 of the study included a

final Bakan test session, check-in of equipment, an

exit questionnaire regarding the study experience and

a cohesiveness questionnaire. This was done to mitigate an end-of-study effect at the end of the previous

week and to allow for data collection on the weekend

following the last shiftwork week.

Physiological Monitor

The Series 2000 MiniloggerTM ambulatory physiological monitor (MiniMitterTM Co., Inc., 20300 Empire Ave., Bend, OR 97701) was used to measure the

inter-beat-interval (IBI) of heart beats, core body temperature, and wrist activity. The monitor was approximately 120 x 65 x 22mm and weighed 125 grams.

Sensors included: 1) a PolarTM chest band for measuring

IBI, 2) a Yellow Springs InstrumentsTM (YSI) flexible

rectal temperature sensor, and 3) a small wrist activity

monitor. All of these channels were direct-wired to the

monitor. A sampling rate of once per minute was used for

temperature and activity. The chest band, an adjustable

elastic band with a rubber section containing electrodes,

was placed around the chest. The temperature sensor was

a flexible tube approximately 36” long. The wrist activity

monitor was approximately 1” square by 0.25” thick and

was worn on the non-dominant wrist like a wristwatch.

Temperature Data

The temperature data were reduced for each participant by computing 1-hour averages for each 24-

hour epoch. All analyses consisted of the final 72

hours of the 3 weeks (Baseline, Shiftwork Week 1, and

Shiftwork Week 2). To control for the effects of

exogenous factors, a custom platform in EXCEL

(Solver) was designed to fit a cosine function to the

averaged temperature data. The function was fit to the

raw data curve by minimizing the sum-of-squares

(ΣSS) representing the sum of point-wise squared

differences between the values of the data and the

curve being fit to each individual data point. In those

few cases where the program failed to fit the curve (i.e.

left a large residual ΣSS), Solver allowed for visual

identification of the mismatch. The amplitude, phase,

and period were then manually adjusted and Solver rerun to obtain an acceptable solution. A more detailed

description can be found in Appendix A.

Salivary Cortisol and Melatonin

Saliva Collection Procedure. Saliva samples were

collected to obtain measurements of cortisol and

melatonin concentrations. Pre-labeled plastic collection vials with cotton rolls were used to collect the

saliva samples. Participants were instructed to rinse

their mouth with cold water 15 minutes before collecting saliva samples and to refrain from eating,

drinking, or chewing gum for 1 hour prior to saliva

collection. Participants placed a cotton roll in their

mouth and held it between their cheek and gums for

up to 3 minutes, then spit any additional saliva into

the collection vial and pushed the cotton roll into the

vial with their tongue to avoid touching it with their

fingers. After capping the vial, samples were placed

directly into a freezer at home or at the laboratory.

Samples were transported from home using

Styrofoam boxes and freezer packs. The samples

were stored at –200

C for long-term storage.

A 24-hour baseline was collected from Thursday to

Friday at the end of week 1 (Days 4-5). Participants

were given pre-labeled salivary collection vials and

instructed to collect their saliva at 0845, 1045, 1400,

1645, 1845, 2200, 0045, 0245, and 0600. They were

given watches programmed with these collection times

so that an auditory alarm would remind them to

collect the sample. The baseline collection times were

chosen to correspond to the laboratory collection

times during the shiftwork weeks (Table 3).

Assay Procedure. Saliva samples were analyzed for

cortisol and melatonin by radioimmunoassay. A

Beckman Gamma 5500 counter was used for the

radioactive counting of all samples. The saliva samples

were thawed at room temperature for approximately

30 minutes. Each sample was placed in a collection

tube and centrifuged for 4 minutes at 3000 g. Detailed procedures for cortisol and melatonin assays are

provided in Appendix B.
6

Design and Data Analysis

Data analyses were conducted utilizing SPSS 10.0

for Windows. The majority of analyses utilized the

General Linear Model for Repeated Measures procedure with the Greenhouse and Geisser (1959) correction for degrees of freedom. Significant main effects

were followed with Tukey’s Honestly Significant

Difference (HSD) tests to compare means. Interactions were followed with appropriate simple effects

analyses. Alpha was set at .05.

The temperature data were analyzed using a 2

(rotation condition) x 3 (week) mixed model design.

A second analysis consisted of a mixed model

ANCOVA using the baseline data as the covariate to

control for the effects of baseline differences on responses to shiftwork.

Two separate analyses were conducted for both

cortisol and melatonin. The initial analyses consisted

of a manipulation check in which the raw scores were

assessed by a series of 3 (week) x 4 (sample) x 2

(rotation condition) mixed-model ANOVAs, where

week and sample were the within-subjects factors, and

rotation condition was the between-subjects factor.

For this analysis, the baseline samples were compared

with their respective, time-locked workweek samples

for the appropriate day (e.g., the first early morning

shift samples of weeks 1 and 2 compared with the

baseline early-morning samples, etc.).

The second set of analyses consisted of comparing the

delta change scores computed by subtracting the baseline

values from the values obtained during the two shiftwork

weeks. To assess individual responses to the tasks, a series

of 2 (week) x 2 (day) x 4 (sample) x 2 (rotation condition)

mixed-model ANOVAs were separately employed for

the morning and afternoon shifts. Since there was

only one midnight shift in both of the workweeks, a

2 (week) x 4 (sample) x 2 (rotation condition) model

was employed.

RESULTS

Core Body Temperature

The results of the ANCOVA for the measures of core

body temperature revealed significant main effects for

rotation condition for both Amplitude, F (1, 21) =

14.62, p <.001 and Acrophase1

, F (1, 21) = 4.43, p < .05.

Amplitude for the counter-clockwise rotation (M = 0.3o

C) was significantly lower than the amplitude for the

clockwise rotation (M = 0.5o

 C). In addition, an 84-

minute delay of the acrophase was found for the counterclockwise condition (M = 1808h) relative to the clockwise

condition ( M = 1644h).

To illustrate these findings, Figure 1 presents the

average temperature values and the cosine, best-fitting

curves for the 72-hr epochs of interest for the clockwise and counter-clockwise rotations. Apparent in the

illustrations is an attenuation of the amplitude of the

curves and an 84-minute delay in the acrophase in the

counter-clockwise rotation, compared with the clockwise rotation.

Table 3

Saliva Sampling Schedule for Shiftwork Weeks

Clockwise Rotation Counter-Clockwise Rotation

Shift1 Collection Times Shift Collection Times

EM1 0600 0845 1045 1400 A1 1400 1645 1845 2200

EM2 0600 0845 1045 1400 A2 1400 1645 1845 2200

A1 1400 1645 1845 2200 EM1 0600 0845 1045 1400

A2 1400 1645 1845 2200 EM2 0600 0845 1045 1400

M 2200 0045 0245 0600 M 2200 0045 0245 0600

1

 EM1=First Early Morning Shift A2 = Second Afternoon Shift

 EM2=Second Early Morning Shift M = Midnight Shift

 A1 = First Afternoon Shift

1

 Acrophase refers to the time of the peak of the temperature rhythm

for a specified period.
7

Figure 1. Temperature Data and Cosine Functions for Clockwise and CounterClockwise Rotation Conditions for All Work Weeks

0000

0800

1600

00000800

1600

0000

0800

1600

0000

37.6

37.4

37.2

37.0

36.8

36.6

36.4

37.8

Temperature (degrees Celcius)

Time of Day

Clockwise Week 1 (Baseline)

0000

0800

1600

0000

0800

1600

0000

0800

1600

0000

37.6

37.4

37.2

37.0

36.8

36.6

36.4

37.8

Temperature (degrees Celcius)

Time of Day

Counter-Clockwise Week 1 (Baseline)

0000

0800

1600

0000

0800

1600

0000

0800

1600

0000

37.6

37.4

37.2

37.0

36.8

36.6

36.4

37.8

Temperature (degrees Celcius)

Time of Day

Clockwise Week 2

0000

0800

1600

0000

0800

1600

0000

0800

1600

0000

37.6

37.4

37.2

37.0

36.8

36.6

36.4

37.8

Temperature (degrees Celcius)

Time of Day

Counter-Clockwise Week 2

0000

0800

1600

0000

0800

1600

0000

0800

1600

0000

37.6

37.4

37.2

37.0

36.8

36.6

36.4

37.8

Temperature (degrees Celcius)

Time of Day

Clockwise Week 3

0000

0800

1600

0000

0800

1600

0000

0800

1600

0000

37.6

37.4

37.2

37.0

36.8

36.6

36.4

37.8

Temperature (degrees Celcius)

Time of Day

Counter-Clockwise Week 3
8

Cortisol

Raw Score Analysis

Sample means for the early morning shifts (EM1

and EM2), afternoon shifts (A1 and A2) and the

midnight shift (M) are presented in Figure 2. The

analysis of the early morning shift data yielded significant main effects for sample for both EM1, F (3, 78)

= 40.68, p < .05 and EM2, F (3, 78) = 43.05, p < .05.

Tukey post hoc comparisons revealed that Sample 1

(0600 h) was significantly higher than Samples 2

(0845 h), 3 (1045 h), or 4 (1400 h) for both earlymorning shifts.

Similarly, significant main effects for sample were

found for A1, F (3, 78) = 41.83, p < .001, and A2, F

(3, 78) = 54.78, p < .001. Tukey post hoc comparisons

indicated that for A1, samples 1 (1400 h) and 2 (1645

h) were significantly higher than samples 3 (1845 h)

and 4 (2200 h), but not different from each other. For

A2, sample 1 (1400 h) was significantly higher than

samples 2 (1645 h), 3 (1845 h), and 4 (2200 h). In

addition, sample 2 (1645 h) was significantly higher

than sample 4 (2200 h).

The omnibus test for the midnight shift resulted in

a significant week by sample interaction, F (6, 150) =

3.09, p < .05. Simple effects analyses of sample at each

level of week revealed significance for all three weeks,

F (4, 100) = 89.78, p < .001, F (4, 100) = 86.25, p <

.001, and F (4, 100) = 64.17, p < .001, respectively.

The post hoc comparisons of the mean differences for

sample at each level of week revealed that sample 4

(0600 h) was significantly higher at each week than

samples 1 (2200 h), 2 (0045 h), and 3 (0245 h). Figure

3 reveals that cortisol levels for the 0600 h sample

during baseline were higher than the two shiftwork

weeks. The least significant difference (LSD) between

the baseline week and the first week of shiftwork was

statistically significant.

Cortisol Delta Change Score Analysis

There were no significant effects on either the early

morning or the afternoon shifts for the change score

analyses for cortisol. Analysis of the change scores for

the midnight shift, however, revealed a significant

main effect for sample, F (3,78) = 4.12, p < .05. Tukey

0

0.1

0.2

0.3

0.4

0.5

2200 0045 0245 0600

Sample Collection Time

Cortisol Level

Baseline

Week 2

Week 3

Figure 3. Cortisol Levels for Midnight Shift (Sample by Week

Interaction)

0

0.1

0.2

0.3

0.4

0.5

0600 0845 1045 1400 1400 1645 1845 2200 2200 0045 0245 0600

Early Morning Shift Afternoon Shift Midnight Shift

Cortisol Level

EM1, A1, M

EM2, A2

Figure 2. Cortisol Levels for Each Shift by Sample Time
9

post hoc comparisons indicated that there was a

significantly greater change from baseline for the 6:00

a.m. sample, when compared with the three earlier

samples (Figure 4).

Melatonin

Raw Score Analysis

Sample means for the (HPA)early morning shifts

(EM1 and EM2), afternoon shifts (A1 and A2), and

the midnight shift (M) are presented in Figure 5. The

analysis of the early morning shift data revealed a

main effect for sample for EM1, F (3, 72) = 49.77, p

< .001 and EM2, F (3, 72) = 28.09, p < .001. Tukey

post hoc comparisons of the mean differences indicated that sample 1 (0600 h) was significantly higher

than samples 2 (0845 h), 3 (1045 h), or 4 (1400 h) for

both shifts. A main effect for rotation condition was

also present for EM1, F (1, 24) = 7.21, p < .05,

indicating that the clockwise group had significantly

higher melatonin levels across all three weeks compared with the counter-clockwise group (Figure 6).

Analysis of melatonin levels for the afternoon and

midnight shifts revealed week by sample interactions

for A1, F (6, 144) = 6.31, p < .01; A2, F (6, 144) =

11.52, p < .001; and M, F (6, 144) = 10.81, p < .001

(Figure 7). The results of simple effects analyses for

sample at each level of week are presented in Table 4.

Tukey post hoc comparisons revealed that for both

afternoon shifts, sample 4 (2200 h) was significantly

higher than samples 1 (1400 h), 2 (1645 h), or 3 (1845

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

2200 0045 0245 0600

Sample Collection Time

Cortisol Change Score

Figure 4. Cortisol Change Score for Midnight Shift Samples

0

1

2

3

4

5

6

7

Clockwise Counter-Clockwise

Rotation Condition

Figure 6. Melatonin Levels for EM1 by

Rotation Condition

0

5

10

15

20

0600 0845 1045 1400 1400 1645 1845 2200 2200 0045 0245 0600

Early Morning Shifts Afternoon Shifts Midnight Shift

Melatonin Level

EM1, A1, M

EM2, A2

Figure 5. Melatonin Levels for Each Shift by Sample Time
10

h). For the midnight shift, post hoc analyses revealed

that during the baseline week, samples 2 (0045 h), 3

(0245 h), and 4 (0600 h) were significantly higher

than sample 1 (2200 h) and that sample 3 (0245 h) was

also higher than samples 2 (0045 h) and 4 (0600 h).

During week 2, samples 2 (0045 h) and 3 (0245 h)

were significantly higher than samples 1 (2200 h) and

4 (0600 h). Finally, during week 3, samples 2 (0045

h), 3 (0245 h), and 4 (0600 h) were significantly

higher than sample 1 (2200 h).

Melatonin Delta Change Score Analysis

There were no significant effects for the melatonin

change scores for the early morning shifts. Analysis of

the afternoon shifts yielded a significant main effect

for sample, F (3, 72) = 11.68, p < .05. Post hoc

comparison of the means indicated that the change for

sample 4 (2200) was significantly greater than samples

1 (1400 h), 2 (1645 h), and 3 (1845 h; Figure 8).

There was also a significant effect for sample on the

midnight shift, F (3, 72) = 31.08, p < 05. The analysis

of the mean differences revealed a significantly greater

change for sample 3 (0245 h) compared with all other

samples, in addition to a greater change for sample 4

(0600 h) compared with Samples 1 (2200 h) and 2

(0045 h; Figure 8).

DISCUSSION

The current investigation compared individuals

working clockwise and counter-clockwise rapidly rotating shifts using three biological markers: core body

temperature, salivary cortisol, and salivary melatonin.

The intention was to determine if either or both of

these shift schedules would elicit changes in the circadian rhythm of temperature or disrupt the secretion

patterns of melatonin and cortisol.

The results of the temperature analysis indicate

that the circadian profile was affected as a function of

shift schedule rotation. The delay in the acrophase

noted in the counter-clockwise group of 84 minutes,

while unexpected in an advancing system, might be

explained as a function of the schedule. In the past, the

counter-clockwise 2-2-1 schedule has been viewed as

a multiply advancing system (Della Rocco & Cruz,

1995), with one advance in the rotation from the

afternoon to the early morning shift and a second

advance from the early morning to the midnight shift.

A closer examination of the properties of the schedule,

however, reveals that the schedule may be better

conceptualized as a mixed system, with one advance

from the afternoon to the early morning shift and one

delayed day exhibited in the early morning to the

Table 4

Simple Effects Analysis of Week by Sample Interaction for Melatonin for Afternoon and

Midnight Shifts

Statistics for A1 Statistics for A2 Statistics for M

Effect df F p-value df F p-value df F p-value

Sample at Week 1 4, 96 10.01 .001 4, 96 37.72 .001 4, 96 10.81 .001

Sample at Week 2 4, 96 6.04 .001 4, 96 32.67 .001 4, 96 4.48 .01

Sample at Week 3 4, 96 3.67 .008 4, 96 30.64 .001 4, 96 3.67 .001

-5

0

5

10

15

20

1400 1645 1845 2200 1400 1645 1845 2200 2200 0045 0245 0600

Afternoon Shift 1 (A1) Afternoon Shift 2 (A2) Midnight Shift (M)

Baseline

Week 2

Week 3

Figure 7. Melatonin Levels for Afternoon and Midnight Shifts by Week and Sample Time
11

midnight shift. That is, individuals work the morning

shift, get 8 hours off then return to work later that

evening for a second 8-hour shift in a 24-hour period.

This prolonged day in the system is more akin to a

delaying system or westward travel. For the counterclockwise group, the early morning shift on the last

day, followed by the quick turn to the midnight shift,

may have been enough to delay the acrophase in

temperature. Such a finding is possible, irrespective of

the nap noted in the counter-clockwise group prior to

the midnight shift (Cruz, Detwiler, Nesthus, &

Boquet, 2002).

The finding that amplitude was attenuated in the

counter-clockwise rotating group is interesting in that

other research supports the idea that amplitude may

be related to individual adaptation to shiftwork

(Czeisler, Moore-Ede, & Coleman, 1982). However,

the results of this research are equivocal, with some

individuals reporting greater adaptability among those

individuals with attenuated amplitude and others

reporting that greater amplitude is indicative of increased shiftwork hardiness (Czeisler et al., 1982).

Therefore, the reduced amplitude in temperature in

the counter-clockwise group could indicate more or

less adaptability to shiftwork.

With respect to HPA activity, the results of the

cortisol analyses indicated that increased levels of

cortisol at the beginning of the early morning shifts

(0600h) and at the end of the midnight shifts (0600h)

can be expected, given the normal early-morning

dump of cortisol consistent with its diurnal pattern

(Guyton & Hall, 2000). Notably, however, the lack of

significant effects for the change score analysis of

cortisol during the early morning and afternoon shifts

indicates that the individual response to the shifts and

the demands of the work environment did not

differentially affect the two groups. Thus, the change

from baseline for both groups was similar, with no

differences noted between the rotation conditions.

This is a somewhat unexpected finding, given the current thinking in the field that would suggest that the

clockwise rotating group would show lower levels of

cortisol.

The raw score analyses of the melatonin data indicate a pattern similar to that of cortisol with the

exception of the first early morning shift. Specifically,

melatonin secretion followed a pattern consistent

with its normal circadian rhythm for both rotation

conditions. The initial early morning shift, however,

revealed a significant effect for rotation condition

with the clockwise group displaying significantly

higher levels compared with the counter-clockwise

group. This difference appears to result at least in part

from higher baseline levels for the clockwise group,

which may be driving the differences noted during the

subsequent two work weeks.

The results of the raw score analyses are further

supported by the observation that no differences were

noted between the rotation conditions in the change

score analysis. The greater changes noted for the later

samples are consistent with diurnally mediated changes

in the system. Thus, no differences were noted in the

responses of either the HPA axis or the melatonin

system with respect to shift rotation.

While few differences were noted with respect to

rotation condition, the preliminary nature of the

current investigation limits the conclusions to be

drawn in a number of ways. First, the participants were

only observed for two weeks of shiftwork; thus, the longterm effects of these two shift systems are not addressed

here. Second, out of convenience to the participants,

only one physiological marker (temperature) was assessed

-6

-5

-4

-3

-2

-1

0

1

1400 1645 1845 2200 2200 0045 0245 0600

Afternoon Shifts Midnight Shift

Figure 8. Melatonin Change Scores for Afternoon and

Midnight Shifts
12

for changes in circadian pattern. While melatonin and

cortisol were assessed during the work periods, they

were not assessed for changes in overall pattern or for

recovery following the workweeks. With this in mind,

there may have been some differences that were not

observed, based upon the current protocol.

As the third paper in a series investigating the

response of individuals to clockwise and counterclockwise, rapidly rotating shift schedules, the findings here reflect minimal differences in biological

markers with respect to the rotation conditions, which

is in line with the findings in the preceding papers

(Cruz et al., 2002; Cruz, Boquet, Detwiler, & Nesthus,

2002). The differences noted for the temperature

rhythm between the rotation conditions, while significant, do not necessarily argue against the counterclockwise rotation on a short-term basis. The absence

of significant effects noted for the response of the

HPA axis and the melatonin system indicate that these

two common markers for shiftwork tolerance were

relatively undisturbed other than for expected diurnal

variations.

In summary, the results from this study support the

assertion by Turek (1986) that clockwise and counterclockwise shift rotations would similarly perturb circadian rhythms. In fact, the results from all the

measures of this study reveal very few significant

differences between the clockwise and counter-clockwise, rapidly rotating shift schedules investigated.

Taken together, these results indicate that counterclockwise schedules currently in use by U.S. air traffic

controllers would not likely be improved simply by

reversing the direction of rotation. Future research

should seek to replicate and extend the findings of this

study by investigating longer shiftwork exposure and

looking more specifically at the entire circadian rhythm

for neuroendocrine measures.

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A1

APPENDIX A

Custom Cosine Function Platform. In order to analyze the temperature data, a brief routine was written

in Microsoft Excel ’97 (1997) using the Solver function that allowed a cosine function to be fit to the raw

temperature data. Solver enables the user to specify minimization of a specified target cell’s value by

automatic manipulation of the values in other specified parameter cells. In this case, Solver’s net effect was

to minimize a sum-of-squares (ΣSS) representing the sum of point-wise squared differences between the

values of the data and the curve being fit to each individual data point. To accomplish this, Solver uses a

Generalized Reduced Gradient (GRG2) nonlinear optimization algorithm developed by Lasdon and

Warren (Excel ’97 Help Files). Conceptually, there are a number of ways of accomplishing this same type

of result (Press, Flannery, Teukolsky, & Vetterling, 1988). Most methods are subject to occasional

minimization failure. This means that ΣSS can get stuck in a local, rather than a global, minimum. In cases

such as these when Solver failed to converge, leaving a large residual, this was visually identified by the

mismatch of the fitting curve and the raw data curve. In the few cases where this occurred, the amplitude,

phase, and the period were manually adjusted so that they were well within the attractor basin of the global

solution on the subsequent re-run of Solver.

One final theoretical problem with this approach was the typical one associated with trying to fit any

periodic function to data sets. In theory, the identical degree of fit is possible with an inverted function

having negative amplitude and being half a period out of phase. To address this problem, the fitting process

produced nothing but positive values for amplitude. For phase, the true value was assumed to be the one

whose absolute value lay closest to zero.

B1

APPENDIX B

Cortisol and Melatonin Assay Procedures

A. Assay for Melatonin

1. Incubation buffer was distributed as described into each of the individual polystyrene tubes. The NSB

(blank) tubes received 500 μL, and the MB (maximum binding) tubes received 400 μL. The standard tubes,

labeled as A through F, received 400 μL of the corresponding standard solution. The samples (400 μL) and

the controls (400 μL) were distributed to their corresponding tubes.

2. 100 μL of the melatonin antibody was delivered to all except the NSB and T (total activity) tubes.

3. 100 μL of the 125I-melatonin tracer was added to all tubes, and all were vortexed. The T tubes were set aside

at this point, since they required no further processing until the final step.

4. All tubes were incubated for 20 hours at 4°C in the refrigerator.

5. 100 μL of the second antibody suspension, continuously being stirred during this step, was added to all assay

tubes (except T tubes). The assay tubes were maintained at 4°C during this process.

6. The tubes, with the added second antibody, were incubated for 15 minutes at 4°C. Then, 1 mL of cold,

distilled water was added to all the tubes (except the T tubes).

7. The tubes were centrifuged for 2 minutes at 2000 x g and 4°C. The supernatants were aspirated and

discarded (except the T tubes), and the precipitates were retained for counting.

8. Finally, the tubes were subjected to counting for 2 minutes each in the Gamma 5500 counter.

B. Assay for Cortisol

1. Each standard, A through F, was diluted 1-in-10 with double-distilled water. The standard tubes, coated

with antibody and labeled A through F, received 200 μL of the corresponding standard. The NSB (blank)

tube was uncoated and received 200 μL of the A standard.

2. 200 μL of each saliva sample was pipetted into antibody coated tubes. This was done in duplicate for each

sample.

3. 1.0 mL of 125I-cortisol was pipetted into every tube and each tube was vortexed for mixing. Two uncoated

tubes, labeled T, received 1.0 mL of 125I-cortisol for obtaining a total radioactive count.

4. The tubes were incubated for 3 hours. After incubation, all tubes, except T tubes, were decanted for 2 to 3

minutes.

5. All tubes were then counted for 1 minute using the Beckman Gamma 5500TM counter.