Disrupted nocturnal melatonin in autism: Association with tumor necrosis factor and sleep disturbances.

Sleep disturbances, abnormal melatonin secretion, and increased inflammation are aspects of autism spectrum disorder (ASD) pathophysiology. The present study evaluated the daily urinary 6-sulfatoxymelatonin (aMT6s) excretion profile and the salivary levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in 20 controls and 20 ASD participants, as well as correlating these measures with sleep disturbances. Although 60% of ASD participants showed a significant night-time rise in aMT6s excretion, this rise was significantly attenuated, compared to controls (P < .05). The remaining 40% of ASD individuals showed no significant increase in nocturnal aMT6s. ASD individuals showed higher nocturnal levels of saliva TNF, but not IL-6. Dysfunction in the initiation and maintenance of sleep, as indicated by the Sleep Disturbance Scale for Children, correlated with night-time aMT6s excretion (r = -.28, P < .05). Dysfunction in sleep breathing was inversely correlated with aMT6s (r = -.31, P < .05) and positively associated with TNF level (r = .42, P < .01). Overall such data indicate immune-pineal axis activation, with elevated TNF but not IL-6 levels associated with disrupted pineal melatonin release and sleep dysfunction in ASD. It is proposed that circadian dysregulation in ASD is intimately linked to heightened immune-inflammatory activity. Such two-way interactions of the immune-pineal axis may underpin many aspects of ASD pathophysiology, including sleep disturbances, as well as cognitive and behavioral alterations.

Monitoring salivary melatonin concentrations in children with sleep disorders using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Melatonin is synthesized in the pineal gland and is an important circadian phase marker, especially in the determination of sleep patterns. Both temporary and permanent abnormal sleep patterns occur in children; therefore, it is desirable to have methods for monitoring melatonin in biological fluids in the diagnosis and treatment of such disorders. OBJECTIVE: The objective of the study is to develop a liquid chromatography-tandem mass spectrometry method for the determination of melatonin in saliva and to apply it to monitoring salivary concentrations in children with sleep disorders. METHODS: A deuterated internal standard (d7-melatonin) was added to a diluted saliva sample (20 µL) in an autosampler vial insert, and 50 µL were injected. Plasticware was strictly avoided, and all glassware was scrupulously cleaned and then baked at 120°C for at least 48 hours to obtain satisfactory performance. Reverse-phase chromatography was performed on a C8 column using a linear gradient elution profile comprising mobile phases A (0.1% aqueous formic acid) and B (15% methanol in acetonitrile containing 0.1% formic acid), pumped at a total flow rate of 0.8 mL/min. The run time was 8 minutes. After atmospheric pressure chemical ionization, mass spectrometric detection was in positive ion mode. Mass detection was by selected reaction monitoring mode with the following mass transitions used for quantification: melatonin, m/z 233.0 → 173.8 and d7-melatonin, m/z 240.0 → 178.3. RESULTS: Linearity (r > 0.999) was established from 3.9 to 1000 pg/mL. Imprecision (coefficient of variation percent) was less than 11%, and accuracy was 100-105% (7.0-900 pg/mL). The method was selective, and the mean (range) ratio of the slopes of calibrations in water to those in daytime saliva samples collected from 10 healthy adult subjects was 0.989 (0.982-0.997), indicating negligible matrix effects. The application of the assay was demonstrated in healthy adults and in children being clinically investigated for sleep disturbances. CONCLUSIONS: A validated liquid chromatography-tandem mass spectrometry method suitable for monitoring salivary melatonin in children with circadian rhythm sleep disorders is reported. The method also has potential application to pediatric population pharmacokinetic studies using sparse sampling of saliva as the biological sample matrix.

Sleep-wake cycles and cognitive functioning in schizophrenia.

BACKGROUND: Irregular sleep-wake cycles and cognitive impairment are frequently observed in schizophrenia, however, how they interact remains unclear. AIMS: To investigate the repercussions of circadian rhythm characteristics on cognitive performance and psychopathology in individuals with schizophrenia. METHOD: Fourteen middle-aged individuals diagnosed with schizophrenia underwent continuous wrist actimetry monitoring in real-life settings for 3 weeks, and collected saliva samples to determine the onset of endogenous melatonin secretion as a circadian phase marker. Moreover, participants underwent multiple neuropsychological testing and clinical assessments throughout the study period. RESULTS: Sleep-wake cycles in individuals with schizophrenia ranged from well entrained to highly disturbed rhythms with fragmented sleep epochs, together with delayed melatonin onsets and higher levels of daytime sleepiness. Participants with a normal rest-activity cycle (objectively determined by high relative amplitude of day/night activity) performed significantly better in frontal lobe function tasks. Stepwise regression analysis revealed that relative amplitude and age represented the best predictors for cognitive performance (Stroop colour-word interference task, Trail Making Test A and B, semantic verbal fluency task), whereas psychopathology (Positive and Negative Syndrome Scale) did not significantly correlate with either cognitive performance levels or the quality of sleep-wake cycles. CONCLUSIONS: Consolidated circadian rhythms and sleep may be a prerequisite for adequate cognitive functioning in individuals with schizophrenia.

Relationship between circadian salivary melatonin levels and sleep-wake behavior in infants.

BACKGROUND: There have been calls for more aggressive intervention for infants with failure in development of a sleep-wake rhythm. If development of the 'biological clock' in infants can be assessed by measuring melatonin, this may provide a useful indicator of the sleep-wake rhythm development. Thus, we investigated relationship between circadian salivary melatonin concentrations and sleep-wake behavioral parameters in infants. METHODS: Sixty-seven mothers who had infants aged 3-15 months were requested to record sleep-wake behavior of their baby for 2 days, and to collect their baby's saliva four times daily in the morning (06:00-09:00 h), noon (11:00-13:00 h), evening (16:00-18:00 h), and night (19:00-22:00 h) for measurement of melatonin concentrations by ELISA. RESULTS: The mean melatonin concentrations of the saliva were: morning 40 ± 4 pg/mL, noon 14 ± 3 pg/mL, evening 15 ± 3 pg/mL, and night 23 ± 4 pg/mL. The melatonin concentrations, at each measurement point, were highest in infants aged 3-5 months, and decreased as age increased. Morning melatonin concentrations showed a negative correlation with nocturnal sleep duration (P<0.05). Increased morning concentrations were related to early waking time (P<0.05). In infants with open air baths on most days, evening and night melatonin concentrations were significantly lower (P<0.05). CONCLUSION: Salivary melatonin concentrations in infants between 06:00 and 22:00 decreased by age, and elevation of morning values may indicate an immature sleep-wake rhythm. Frequent open air baths may contribute to decreased melatonin levels.

Clinical efficacy of dim light melatonin onset testing in diagnosing delayed sleep phase syndrome.

BACKGROUND: Delayed Sleep Phase Syndrome (DSPS) arises from biological clock desynchrony and accounts for 10% of chronic insomnia patients. Currently DSPS is diagnosed based on sleep/wake cycle disruptions rather than examining the underlying biological clock alterations. The objective of the study was to determine the sensitivity and specificity of the Dim Light Melatonin Onset (DLMO) Test in diagnosing DSPS in a clinical setting. METHODS: Fifty-six patients (mean age 28 years) symptomatic of DSPS participated in the study. Following an initial assessment of DSPS using sleep diaries, participants underwent two consecutive nights of polysomnography (PSG), with an imposed sleep period on the second night to demonstrate the delay in the timing of habitual sleep period and to thereby confirm DSPS. Circadian phase delays were also measured using melatonin secretion profiles, and the efficacy of diagnosing DSPS using DLMO was compared to using sleep diaries and PSG. Melatonin secretion was assayed for each individual by ELISA using saliva samples. RESULTS: Main outcome measures included the time of melatonin secretion onset, clinical sensitivity and specificity of the DLMO test. The time of melatonin secretion onset was significantly delayed in DSPS patients. Clinical sensitivity and specificity of the DLMO test in diagnosing DSPS were 90.3% and 84.0%, respectively. CONCLUSIONS: The DLMO test is an accurate tool for differentiating between sleep disorder patients with or without underlying circadian rhythm disruption. It is effective for phase typing DSPS patients in a clinical setting.

Salivary melatonin onset in youth at familial risk for bipolar disorder.

Melatonin secretion and polysomnography (PSG) were compared among a group of healthy adolescents who were at high familial risk for bipolar disorder (HR) and a second group at low familial risk (LR). Adolescent participants (n = 12) were a mean age 14 ± 2.3 years and included 8 females and 4 males. Saliva samples were collected under standardized condition light (red light) and following a 200 lux light exposure over two consecutive nights in a sleep laboratory. Red Light Melatonin onset (RLMO) was defined as saliva melatonin level exceeding the mean of the first 3 readings plus 2 standard deviations. Polysomnography was also completed during each night. HR youth, relative to LR, experienced a significantly earlier melatonin onset following 200 lux light exposure. Polysomnography revealed that LR youth, relative to HR, spent significantly more time in combined stages 3 and 4 (deep sleep) following red light exposure. Additionally, regardless of the group status (HR or LR), there was no significant difference in Red Light Melatonin Onset recorded at home or in the laboratory, implying its feasibility and reliability.

The cortisol awakening response: a pilot study on the effects of shift work, morningness and sleep duration.

This study concerned the possible influence of experimental shift work, morningness and sleep length on the cortisol awakening response (CAR). Eight morning-oriented (MT) and eight evening-oriented (ET) healthy young men (19-27 years) slept after three consecutive day shifts during the night and after three consecutive night shifts during the day in the laboratory. Salivary cortisol concentrations were ascertained after each sleep period upon awakening and half an hour later, half-hourly during work shifts, and hourly during two 24-h periods, after the three day shift/night sleep sequences and after the three night shift/day sleep sequences. Statistical analyses considered the temporal position of sleep (night, day), the succession of sleep periods, the diurnal type and the polysomnographically verified total sleep time. The CAR was significantly smaller after day than after night sleep and increased significantly with total sleep time in ET. MT had moderately higher cortisol concentrations upon awakening than ET probably because they wake up at a later time of their circadian rhythm. But neither the CARs nor the cortisol concentrations during the following work shifts or during the 24h profiles were different in both diurnal types. The cortisol concentrations during work shifts correlated significantly with the previous post-awakening concentrations in MT but not in ET. Due to the small samples further studies are needed.

No effects of repeated forced wakings during three consecutive nights on morning cortisol awakening responses (CAR): a preliminary study.

OBJECTIVE: The cortisol awakening response (CAR) is considered a reliable measure for the acute reagibility of the hypothalamus-pituitary-adrenal (HPA) axis. Whether repeated nightly awakenings at different times during the night also stimulate the HPA-axis and whether, consequently, the CAR is altered has not been tested, so far. We aimed to investigate whether three experimentally induced awakenings during three consecutive nights would be associated with HPA-axis stimulation and an altered morning CAR. METHODS: The study sample consisted of 13 healthy adult women who were waken up three times in each of three consecutive nights. Cortisol levels were measured immediately and 15 min after each awakening in the night and in the morning, respectively. Also, the morning CARs after three nights of undisturbed sleep were assessed. RESULTS: A significant difference between night time cortisol responses to awakening and the morning CAR was found. While cortisol levels during the first half of the night did not rise significantly after awakening in the night, some reactivity was seen during awakenings in the very early morning hours, and pronounced awakening responses were observed in the morning before getting out of bed. Interestingly, the morning CAR after disturbed sleep did not differ from the morning CAR following undisturbed sleep. CONCLUSION: In healthy female individuals, the morning CAR appears to be unchanged even if sleep was repeatedly interrupted by forced wakings.

Circadian cortisol profiles and psychological self-reports in shift workers with and without recent change in the shift rotation system.

Cortisol profiles including the cortisol rise in the first hour after awakening (CAR) were assessed during shift work and days off (eight saliva samples per shift). Participants were 102 healthy permanent day and night shift workers (comparison groups) and former permanent day and night shift workers after implementation of a new fast-forward rota including morning, evening, and night shifts. Results show that the CAR is detectable in day as well as night shifts. In permanent night workers cortisol profiles appear to be blunted during night work and days off. However, circadian cortisol profiles are not disturbed in former night workers who recently switched to the fast rotating shift schedule. In contrast, implementation of night work in former day workers seems to lead to initially blunted cortisol profiles that normalize after a short adjustment period. Results of a psychological assessment including exhaustion, chronic stress, effort-reward imbalance, and ratings of sleep quality and sleep length are also presented.

Cortisol and wake time in nursing home residents with behavioral symptoms of dementia.

Alterations in sleep and behavioral symptoms are consistently reported among nursing home residents with dementia. Disregulation in the hypothalamic-pituitary-adrenal axis (HPA), indexed by basal cortisol levels, offers one explanation. The purpose of this study is to examine the relationship between wake time and cortisol slope in residents with behavioral symptoms. The study included 27 residents aged 71 to 84 years with dementia and behavioral symptoms. Using a within-subject longitudinal design, the researchers documented wake time and collected saliva samples for 4 consecutive days upon waking and at 30 min, 6 hr, and 12 hr after waking. Within-person cortisol slopes were categorized into zero-positive and negative slopes. The zero-positives (35%) exhibited an earlier wake time than the negatives (65%). These preliminary results suggest both a relationship between wake time and HPA diurnal profile and an association between the sleep-wake cycle and cortisol secretion among nursing home residents with dementia.

Circadian Phase and Phase Angle Disorders in Primary Insomnia.

Objectives: We aimed to identify the prevalence of circadian phase and phase angle abnormalities in patients with insomnia. Methods: We conducted a cross-sectional, multicenter study at three sleep laboratories in the United States and Australia. Patients with insomnia and healthy control participants completed a sleep log for 7 days. Circadian phase was assessed from salivary dim light melatonin onset (DLMO) time during a 12-hour laboratory visit. Results: Seventy-nine patients meeting the Research Diagnostic Criteria for Primary, Psychophysiological, Paradoxical, and/or Idiopathic Childhood Insomnia (46 females, 35.5 ± 12.3 years [M ± SD]) and 21 controls (14 females, 34.4 ± 11.8 years). As compared to controls, patients with insomnia tried to initiate sleep on average at the same clock time (24:17 ± 1:17 hours vs. 24:13 ± 1:30 hours, respectively; p = .84) but had a later average DLMO times (20:56 ± 1:55 hours, 18:17-01:21 vs. 22:02 ± 2:02 hours, 17:11-04:52, respectively; p = .04). Consequently, patients with insomnia slept at an earlier circadian phase than controls (phase angle, bedtime-DLMO 2:13 hours (± 1:43) vs. 3:10 hours (± 1:08), respectively; p = .008), of whom 10% tried to sleep at or before DLMO (compared to 0 controls), and 22% tried to sleep before or within 1 hour after DLMO (compared to 6% of controls). Conclusions: A substantial proportion (10%-22%) of patients with insomnia initiate sleep at too early a circadian phase, implicating a circadian etiology for their insomnia. Outpatient circadian phase assessments should be considered to improve differential diagnoses in insomnia and to inform the development of appropriately timed circadian-based treatments.

Estimating dim light melatonin onset (DLMO) phase in adolescents using summer or school-year sleep/wake schedules.

STUDY OBJECTIVES: This analysis examined associations between the salivary dim light melatonin onset (DLMO) phase and self-selected sleep/ wake schedules in groups of children and adolescents during summer vacation and during the school year to determine the degree to which sleep/wake patterns can estimate salivary DLMO phase. DESIGN AND SETTING: Participants slept at home on self-selected schedules for 5 consecutive nights and reported their bedtime and wake-up time via daily telephone messages. Salivary melatonin was sampled in the laboratory on one evening every 30 minutes in dim light (< 50 lux) to determine DLMO phase. Within group bivariate regressions between sleep pattern measures (bedtime, wake-up time, and midsleep time) and DLMO phase were computed. PARTICIPANTS: One group, ages 9 to 17 years (mean age = 12.5, SD = 2.3 years, 74 males, 75 females) contributed 149 DLMO phase and sleep/wake pattern measures while on a school year schedule ("school group"). A separate group, ages 9 to 16 years (mean age = 13.1, SD = 1.3 years, 30 males, 29 females) contributed 59 DLMO phase and sleep/wake pattern measures while on a summer schedule ("summer group"). RESULTS: Bedtime, midsleep time, and wake-up time were positively correlated with DLMO phase in both groups. Although all correlation coefficients for the summer group were statistically greater compared to the school group, the regression equations predicted DLMO phase within +/- 1 hour of the measured DLMO phase in approximately 80% for both groups. CONCLUSIONS: DLMO phase can be estimated using self-selected sleep/wake patterns during the school year or summer vacation in healthy children and adolescents.

Nocturnal Melatonin Profiles in Patients with Delayed Sleep-Wake Phase Disorder and Control Sleepers.

A significant delay in the timing of endogenous circadian rhythms has been associated with delayed sleep phase disorder (DSPD). More recently, other mechanisms have also been proposed to account for this disorder. To further explore the etiology of DSPD, the present study compared nocturnal melatonin profiles of 26 DSPD patients (18 males, 8 females; age, 21.73 ± 4.98 years) and 17 normally timed good sleepers (10 males, 7 females; age, 23.82 ± 5.23 years) in a time-free, dim-light (<10 lux) laboratory environment. A 30-h modified constant routine with alternating 20-min sleep opportunities and 40 min of enforced wakefulness was used to measure the endogenous melatonin circadian rhythm. Salivary melatonin was sampled half-hourly from 1820 h to 0020 h and then hourly from 0120 h to 1620 h. DSPD patients had significantly later timed melatonin profiles that were delayed by approximately 3 h compared to normal sleepers, and there were no notable differences in the relative duration of secretion between groups. However, melatonin secretion between dim-light melatonin onset (DLMO) and acrophase was less prominent in DSPD patients compared to good sleepers, who showed a more acute initial surge of melatonin following the DLMO. Although the regulatory role of melatonin is unknown, abnormal melatonin profiles have been linked to psychiatric and neurological disorders (e.g., major depression, obsessive compulsive disorder, Parkinson disease). These results therefore suggest that in addition to a delayed endogenous circadian rhythm, a diminished initial surge of melatonin secretion following DLMO may contribute to the etiology of DSPD.

The biological clock modulates the human cortisol response in a multiplicative fashion.

Human cortisol levels follow a clear circadian rhythm. We investigated the contribution of alternation of sleep and wakefulness and the circadian clock, using forced desynchrony. Cortisol levels were best described by a multiplication of a circadian and a wake-time component. The human cortisol response is modulated by circadian phase. Exposure to stress at an unnatural phase, as in shift work, is predicted to result in abnormal cortisol levels. Health of shift workers may therefore improve when stress is reduced at times when the clock produces high stress sensitivity.

Influences of early shift work on the diurnal cortisol rhythm, mood and sleep: within-subject variation in male airline pilots.

We aimed to investigate how early and late work shifts influenced the diurnal cortisol rhythm using a within-subjects study design. Participants were 30 healthy male non-smoking pilots, mean age 39.4, employed by a short-haul airline. The standard rotating shift pattern consisted of 5 early shifts (starting before 0600 h), followed by 3 rest days, 5 late shifts (starting after 1200 h) and 4 rest days. Pilots sampled saliva and completed subjective mood ratings in a logbook 6 times over the day on two consecutive early shift days, two late days and two rest days. Sampling was scheduled at waking, waking+30 m, waking+2.5 h, waking+8 h, waking+12 h and bedtime. Waking time, sleep duration, sleep quality and working hours were also recorded. Cortisol responses were analysed with repeated measures analysis of variance with shift condition (early, late, rest) and sample time (1-6) as within-subject factors. Early shifts were associated with a higher cortisol increase in response to awakening (CAR(i)), a greater total cortisol output over the day (AUC(G)) and a slower rate of decline over the day than late shifts or rest days. Early shifts were also associated with shorter sleep duration but co-varying for sleep duration did not alter the effects of shift on the cortisol rhythm. Both types of work shift were associated with more stress, tiredness and lower happiness than rest days, but statistical adjustment for mood ratings did not alter the findings. Early shift days were associated with significantly higher levels of circulating cortisol during waking hours than late shifts or rest days.

Effects of filtering visual short wavelengths during nocturnal shiftwork on sleep and performance.

Circadian phase resetting is sensitive to visual short wavelengths (450-480 nm). Selectively filtering this range of wavelengths may reduce circadian misalignment and sleep impairment during irregular light-dark schedules associated with shiftwork. We examined the effects of filtering short wavelengths (<480 nm) during night shifts on sleep and performance in nine nurses (five females and four males; mean age ± SD: 31.3 ± 4.6 yrs). Participants were randomized to receive filtered light (intervention) or standard indoor light (baseline) on night shifts. Nighttime sleep after two night shifts and daytime sleep in between two night shifts was assessed by polysomnography (PSG). In addition, salivary melatonin levels and alertness were assessed every 2 h on the first night shift of each study period and on the middle night of a run of three night shifts in each study period. Sleep and performance under baseline and intervention conditions were compared with daytime performance on the seventh day shift, and nighttime sleep following the seventh daytime shift (comparator). On the baseline night PSG, total sleep time (TST) (p < 0.01) and sleep efficiency (p = 0.01) were significantly decreased and intervening wake times (wake after sleep onset [WASO]) (p = 0.04) were significantly increased in relation to the comparator night sleep. In contrast, under intervention, TST was increased by a mean of 40 min compared with baseline, WASO was reduced and sleep efficiency was increased to levels similar to the comparator night. Daytime sleep was significantly impaired under both baseline and intervention conditions. Salivary melatonin levels were significantly higher on the first (p < 0.05) and middle (p < 0.01) night shifts under intervention compared with baseline. Subjective sleepiness increased throughout the night under both conditions (p < 0.01). However, reaction time and throughput on vigilance tests were similar to daytime performance under intervention but impaired under baseline on the first night shift. By the middle night shift, the difference in performance was no longer significant between day shift and either of the two night shift conditions, suggesting some adaptation to the night shift had occurred under baseline conditions. These results suggest that both daytime and nighttime sleep are adversely affected in rotating-shift workers and that filtering short wavelengths may be an approach to reduce sleep disruption and improve performance in rotating-shift workers.

Evaluation of salivary melatonin measurements for Dim Light Melatonin Onset calculations in patients with possible sleep-wake rhythm disorders.

BACKGROUND: Dim Light Melatonin Onset (DLMO) can be calculated within a 5-point partial melatonin curve in saliva collected at home. We retrospectively analyzed the patient melatonin measurements sample size of the year 2008 to evaluate these DLMO calculations and studied the correlation between diary or polysomnography (PSG) sleep onset and DLMO. METHODS: Patients completed an online questionnaire. If this questionnaire pointed to a possible Delayed Sleep Phase Disorder (DSPD), saliva collection devices were sent to the patient. Collection occurred at 5 consecutive hours. Melatonin concentration was measured with a radioimmunoassay and DLMO was defined as the time at which the melatonin concentration in saliva reaches 4 pg/mL. Sleep onset time was retrieved from an online one-week sleep diary and/or one-night PSG. RESULTS: A total of 1848 diagnostic 5-point curves were obtained. DLMO could be determined in 76.2% (n=1408). DLMO significantly differed between different age groups and increased with age. Pearson correlations (r) between DLMO and sleep onset measured with PSG or with a diary were 0.514 (p=<0.001, n=54) and 0.653 (p=0.002, n=20) respectively. CONCLUSION: DLMO can be reliably measured in saliva that is conveniently collected at home. DLMO correlates moderately with sleep onset.

The normalization of the cortisol awakening response and of the cortisol shift profile across consecutive night shifts--an experimental study.

This study tested the hypothesis that the cortisol awakening response (CAR) and the cortisol shift profile normalize with successive night shifts due to the shift of the circadian system. 18 students (9 women, 9 men, 19-29 years), worked first four consecutive morning- and then four consecutive night shifts. Each work shift was preceded by an 8-h sleep opportunity meaning that the sleep-activity cycle was advanced by 8 h. The advance of the circadian system was promoted by a 2-h bright light pulse at the end of each night shift and quantified by 24-h phase assessment procedures (PA) before and after the four day shifts and again after the four night shifts. Saliva samples were taken 0, 15, 30, 45, and 60 min post-awakening, hourly during each work shift and each PA. During the night shift sequence, the CAR, indicated by the area under curve with respect to increase (AUC(I)), increased gradually across the 4-day sleep periods. Baseline levels were reached after 3 days in men and 4 days in women. The increase of the CAR was associated with a gradually increasing decline of cortisol levels during the night shifts. This adjustment was--at least not only--related to the advance of the circadian system which was 5 h. A contributor to the increase of the CAR might be the anticipation of the upcoming demands of the following work shifts.

The effect of shift rotation on variations of cortisol, fatigue and sleep in sound engineers.

The aim was to study the effect of rotation direction on the time-of-day variations of cortisol, fatigue and sleep in sound engineers broadcasting in a life show. The salivary cortisol and ratings of stress, sleepiness and fatigue were followed at three hour intervals in 25 sound engineers: 13 working very fast forward-rotating shifts and 12 working very fast backward-rotating shifts. Cortisol was assessed with radioimmunological kits. The participants reported for stress symptoms and filled sleep diary. Cortisol retained the typical diurnal pattern. The rotation direction interacted with the shift significantly and as a result higher cortisol values during the morning and night shifts in the backward rotating group were found as well as worse quality of sleep. Higher salivary cortisol during morning and night shifts and worse quality of sleep in engineers working very fast backward-rotating shifts may be an indication for insufficient recovery.