Circadian rhythm of circulating levels of the endocannabinoid 2-arachidonoylglycerol.

CONTEXT: The endocannabinoid (eCB) system is involved in the regulation of food intake and of peripheral metabolism. Although the cross talk between energy metabolism and the circadian system is well documented, little is known about a potential circadian modulation of human eCB activity. OBJECTIVE: The objective of the study was to define the 24-hour profile of circulating levels of the most abundant endogenous ligand of the CB1 receptor, 2-arachidonoylglycerol (2-AG), in healthy young nonobese adults studied under controlled bedtime, dietary, and activity conditions. METHODS: Fourteen subjects participated in this 4-day laboratory study with fixed light-dark cycles, standardized meals, and bedtimes. Sleep was recorded each night. On the third day, blood sampling at 15- to 30-minute intervals began at 9:30 pm and continued for 24 hours. Cortisol, leptin, and ghrelin were assayed on all samples, whereas the levels of 2-AG and its structural analog, 2-oleoylglycerol (2-OG), were measured at 60-minute intervals. RESULTS: All participants exhibited a large circadian variation of 2-AG serum concentrations with a nadir around midsleep, coincident with the middle of the overnight fast. Levels of 2-AG increased continually across the morning, peaking in the early to midafternoon. Peak values represented, on average, a nearly 3-fold increase above nocturnal nadir levels. Concentrations of 2-OG followed a similar pattern, although with a shorter morning increase and lower amplitude. CONCLUSIONS: The findings demonstrate that activity of the eCB system is profoundly modulated by circadian rhythmicity and suggest that its impact on the regulation of food intake is suppressed during sleep and is maximal during early to midafternoon.

Adverse effects of two nights of sleep restriction on the hypothalamic-pituitary-adrenal axis in healthy men.

CONTEXT: Insufficient sleep is associated with increased cardiometabolic risk. Alterations in hypothalamic-pituitary-adrenal axis may underlie this link. OBJECTIVE: Our objective was to examine the impact of restricted sleep on daytime profiles of ACTH and cortisol concentrations. METHODS: Thirteen subjects participated in 2 laboratory sessions (2 nights of 10 hours in bed versus 2 nights of 4 hours in bed) in a randomized crossover design. Sleep was polygraphically recorded. After the second night of each session, blood was sampled at 20-minute intervals from 9:00 am to midnight to measure ACTH and total cortisol. Saliva was collected every 20 minutes from 2:00 pm to midnight to measure free cortisol. Perceived stress, hunger, and appetite were assessed at hourly intervals by validated scales. RESULTS: Sleep restriction was associated with a 19% increase in overall ACTH levels (P < .03) that was correlated with the individual amount of sleep loss (rSp = 0.63, P < .02). Overall total cortisol levels were also elevated (+21%; P = .10). Pulse frequency was unchanged for both ACTH and cortisol. Morning levels of ACTH were higher after sleep restriction (P < .04) without concomitant elevation of cortisol. In contrast, evening ACTH levels were unchanged while total and free cortisol increased by, respectively, 30% (P < .03) and 200% (P < .04). Thus, the amplitude of the circadian cortisol decline was dampened by sleep restriction (-21%; P < .05). Sleep restriction was not associated with higher perceived stress but resulted in an increase in appetite that was correlated with the increase in total cortisol. CONCLUSION: The impact of sleep loss on hypothalamic-pituitary-adrenal activity is dependent on time of day. Insufficient sleep dampens the circadian rhythm of cortisol, a major internal synchronizer of central and peripheral clocks.

Sleep disturbances and insulin resistance.

The causes and risk factors of insulin resistance remain insufficiently understood. After taking into account the important roles of adiposity, age, sex and race/ethnicity, up to 50% of the individual variability in insulin resistance remains unexplained. In recent years, evidence has accumulated to support a role for sleep disturbances, including insufficient sleep, poor sleep quality and insomnia, and obstructive sleep apnoea, as independent risk factors for the development and exacerbation of insulin resistance. The present review summarizes the evidence. We will start with a brief introduction to sleep and its disorders and then examine in succession the role of the three major types of sleep disturbances of modern society, namely insufficient sleep, poor sleep quality and/or insomnia and obstructive sleep apnoea. Insulin resistance is a hallmark of the polycystic ovary syndrome, the most common endocrine pathology in women, and the last section of this review will discuss the role of obstructive sleep apnoea in the insulin resistance and metabolic disturbances of polycystic ovary syndrome.

Sex differences in the neuroendocrine response to short-term fasting in rhesus macaques.

When energy intake is restricted in mammals, there are neuroendocrine adjustments in the secretion of reproductive and metabolic hormones to reallocate energy for vital functions. In the present study, we investigated whether there were differences in the luteinising hormone (LH), growth hormone (GH) and cortisol responses to a 48-h fast in adult gonad-intact male and female rhesus macaques. In both male and female macaques, blood glucose levels were significantly lower in fasted than in control studies, and levels were higher in males than in females. Male rhesus monkeys had significantly lower (P < 0.01) mean serum LH levels after a 48-h fast than under fed conditions and this was attributable primarily to a decrease in the amount of LH released during each secretory episode. In fasted females, serum LH levels were significantly greater (P < 0.05) than during the fed conditions but no differences were found in pulse amplitude or in the number of pulses. Almost twice as many GH pulses were observed in both males and females during fasting but there was no difference in either mean serum GH levels or pulse amplitude between control and fasted studies. A typical diurnal profile in cortisol levels was observed in both sexes and both experimental conditions. Under control conditions, male macaques released less cortisol than females, and although fasting increased mean cortisol levels in both males and females, only the males shown a significant rise over levels observed in control studies. The changes in plasma LH and cortisol levels in fasted rhesus macaques are similar to those observed in humans and suggest that gonadotrophin and corticotrophin secretion are more resistant to short-term energy deprivation in female than in male primates.

[Impact of sleep debt on physiological rhythms]. / Impact d'une dette de sommeil sur les rythmes physiologiques.

Sleep loss due to voluntary bedtime curtailment has become a hallmark of modern society. Even though sleep deprivation in rodents has been shown to result in death, it was until a few years ago thought that sleep loss results in increased sleepiness and decreased cognitive performance but has little or no adverse effects on human health. We measured sleep and 24-hour hormonal profiles in 11 healthy young males after 6 days of sleep restriction (4-hour bedtime) and after 6 days of sleep recovery (12-hour bedtime). At the end of sleep restriction, we observed reduced amounts of slow wave sleep (SWS) and rapid eye movement (REM) sleep and an alteration in the temporal distribution of these sleep stages, i.e. an increased pressure for REM sleep at the beginning of the sleep period and a decrease in the amount of slow wave activity (SWA) during the first sleep cycle. These later abnormalities are usually observed in depression. In addition, numerous alterations in the 24-hour hormonal profiles were observed in the state of sleep debt. The amount of melatonin secreted was reduced because of a delay in the onset of the nocturnal secretion and a reduction in the value of the acrophase. If the overall 24-hour cortisol profile was preserved, sleep restriction was associated with increased cortisol levels in late afternoon and evening hours and the duration of the quiescent period was reduced. The 24-hour mean TSH levels were reduced and the nocturnal TSH elevation was markedly dampened, most likely as a result of elevated levels of thyroid hormones. The acrophase of the 24-hour leptin profile occurred earlier, the amplitude of the rhythm and the overall mean levels were reduced. The nocturnal elevation of prolactin levels was abrupt but of short duration and the 24-hour mean levels were decreased. A pulse of growth hormone occurred prior to sleep onset, therefore affecting SWA distribution at the beginning of the sleep period. Since these alterations are qualitatively and quantitatively similar to those observed during aging and sometimes during depression, a state of sleep debt, as is experienced by a substantial fragment of the population in modern societies, is likely to increase the severity of depression and widespread age-related chronic conditions such as obesity, diabetes and hypertension.

Transition from dim to bright light in the morning induces an immediate elevation of cortisol levels.

The only well documented effect of light exposure on endocrine function is the suppression of nocturnal melatonin. Bright light exposure has behavioral effects, including the alleviation of sleepiness during nocturnal sleep deprivation. The present study examines the effects of bright light on the profiles of hormones known to be affected by sleep deprivation (TSH) or involved in behavioral activation (cortisol). Eight healthy men participated each in three studies involving 36 h of continuous wakefulness. In one study, the subjects were exposed to constant dim light (baseline). In the two other studies, dim light exposure was interrupted by a 3-h period of bright light exposure either from 0500-0800 h (early morning study) or from 1300-1600 h (afternoon study). Blood samples were obtained every 15 min for 24 h to determine melatonin, cortisol, and TSH concentrations. Alertness was estimated by the number of lapses on two computerized vigilance-sensitive performance tasks. The early morning transition from dim to bright light suppressed melatonin secretion, induced an immediate, greater than 50% elevation of cortisol levels, and limited the deterioration of alertness normally associated with overnight sleep deprivation. No effect was detected on TSH profiles. Afternoon exposure to bright light did not have any effect on either hormonal or behavioral parameters. The data unambiguously demonstrate an effect of light on the corticotropic axis that is dependent on time of day.

A benzodiazepine hypnotic facilitates adaptation of circadian rhythms and sleep-wake homeostasis to an eight hour delay shift simulating westward jet lag.

STUDY OBJECTIVES: To determine whether appropriately timed administration of a short-acting benzodiazepine hypnotic, which has proven effective in an animal model of jet lag, also facilitates adaptation of circadian rhythmicity and sleep-wake homeostasis in a human model of jet lag. DESIGN: Subjects participated in two double-blind, placebo-controlled studies of adaptation to an 8-hr delay shift of sleep-wake and dark-light cycles simulating westward travel. Each 9-day laboratory study began with a 3-day habituation period followed by a 24-hr study to obtain basal hormonal and sleep profiles (23:00-07:00). Subjects were then kept awake until 07:00 the next day and slept in darkness 07:00-15:00 for the next five 24-hr spans post-shift. SETTING: N/A. PARTICIPANTS: 6 normal, healthy men 24-31 years of age. INTERVENTIONS: Oral Triazolam (0.5 mg) or placebo given at 04:00 before the first shifted sleep/dark period (3 hours before bedtime) and at 07:00 (at bedtime) on days 2-5 post-shift. MEASUREMENTS AND RESULTS: Sleep recordings and 24-hr cortisol and growth hormone profiles were obtained at baseline and on the first, third, and fifth days post-shift. Global measures of treatment efficacy were calculated for multiple endpoints representing circadian rhythmicity and sleep-wake homeostasis. With placebo, the shift induced disturbances of sleep and hormonal secretion, and a gradual re-entrainment of circadian rhythmicity. Triazolam significantly facilitated adaptation by accelerating re-entrainment of circadian rhythms (chronobiotic effect) and normalizing markers of sleep/wake homeostasis (hypnotic effect). CONCLUSIONS: Appropriately timed administration of a benzodiazepine hypnotic appears to facilitate the adaptation of both circadian rhythmicity and sleep-wake homeostasis to a shifted dark/sleep cycle. Compounds with combined chronobiotic/hypnotic properties may be useful in conditions of jet lag or night work.

Depression and sleep disorders: clinical relevance, economic burden and pharmacological treatment.

A wide range of studies have been published over the past two decades that involve the intersection of sleep EEG, insomnia, psychiatric illness (especially depressive disorders) and psychopharmacology. Much of value has been discovered, but there have also been false starts and contradictory results. There is in fact strong evidence that insomnia is associated with medical and psychiatric illness and that the sleepiness associated with insomnia is the cause of many accidents. Thus, the direct (visits to doctors, cost of sleeping medication, complications from use of these medications) and indirect (accidents, quality of life) costs of insomnia are enormous and constitute a major public health problem in the industrialized countries. Believing that it is now timely to assess the state of this important research area, a consensus conference was convened on June 26-28, 1998, in Porto Cervo (Italy) to attempt to clarify the important issues and findings on the clinical effect of the different classes of antidepressant drugs on sleep quality in depression. The participants' consensus on some of the main topics is presented with the hope that this discussion and analysis will contribute to productive research in this important field.

Interrelationships between growth hormone and sleep.

In healthy young adults, the 24-hour profile of plasma growth hormone (GH) levels consists of stable low levels abruptly interrupted by bursts of secretion. In normal women, daytime GH secretory pulses are frequent. However, in normal men, a sleep-onset-associated pulse is generally the major or even the only daily episode of active secretion. Extensive evidence indicates the existence of a consistent relationship between slow-wave (SW) sleep and increased GH secretion. There is a linear relationship between the amount of SW sleep (measured by either visual scoring or spectral analysis of the EEG) and the amount of concomitant GH secretion. During ageing, SW sleep and GH secretion decrease exponentially and with the same chronology. Pharmacological stimulation of SW sleep results in increased GH release, and compounds that increase SW sleep may therefore represent a novel class of GH secretagogues.

Adaptation of the 24-h growth hormone profile to a state of sleep debt.

In normal men, the majority of GH secretion occurs in a single large postsleep onset pulse that is suppressed during total sleep deprivation. We examined the impact of semichronic partial sleep loss, a highly prevalent condition, on the 24-h growth hormone profile. Eleven young men were studied after six nights of restricted bedtimes (0100-0500) and after 7 nights of extended bedtimes (2100-0900). Slow-wave sleep (SWS) was estimated as the duration of stages III and IV. Slow-wave activity (SWA) was calculated as electroencephalogram power density in the 0.5- to 3-Hz frequency range. During the state of sleep debt, the GH secretory pattern was biphasic, with both a presleep onset "circadian" pulse and a postsleep onset pulse. Postsleep onset GH secretion was negatively related to presleep onset secretion and tended to be positively correlated with the amount of concomitant SWA. When sleep was restricted, both SWS and SWA were increased during early sleep. Unexpectedly, the increase in SWA affected the second, rather than the first, SWA cycle, suggesting that presleep onset GH secretion may have limited SWA in the first cycle, possibly via an inhibition of central GH-releasing hormone activity. Thus neither the GH profile nor the distribution of SWA conformed with predictions from acute sleep deprivation studies, indicating that adaptation mechanisms are operative during chronic partial sleep loss.

Age-related changes in slow wave sleep and REM sleep and relationship with growth hormone and cortisol levels in healthy men.

CONTEXT: In young adults, sleep affects the regulation of growth hormone (GH) and cortisol. The relationship between decreased sleep quality in older adults and age-related changes in the regulation of GH and cortisol is unknown. OBJECTIVE: To determine the chronology of age-related changes in sleep duration and quality (sleep stages) in healthy men and whether concomitant alterations occur in GH and cortisol levels. DESIGN AND SETTING: Data combined from a series of studies conducted between 1985 and 1999 at 4 laboratories. SUBJECTS: A total of 149 healthy men, aged 16 to 83 years, with a mean (SD) body mass index of 24.1 (2.3) kg/m( 2), without sleep complaints or histories of endocrine, psychiatric, or sleep disorders. MAIN OUTCOME MEASURES: Twenty-four-hour profiles of plasma GH and cortisol levels and polygraphic sleep recordings. RESULTS: The mean (SEM) percentage of deep slow wave sleep decreased from 18.9% (1.3%) during early adulthood (age 16-25 years) to 3.4% (1.0%) during midlife (age 36-50 years) and was replaced by lighter sleep (stages 1 and 2) without significant increases in sleep fragmentation or decreases in rapid eye movement (REM) sleep. The transition from midlife to late life (age 71-83 years) involved no further significant decrease in slow wave sleep but an increase in time awake of 28 minutes per decade at the expense of decreases in both light non-REM sleep (-24 minutes per decade; P<.001) and REM sleep (-10 minutes per decade; P<.001). The decline in slow wave sleep from early adulthood to midlife was paralleled by a major decline in GH secretion (-372 microg per decade; P<.001). From midlife to late life, GH secretion further declined at a slower rate (-43 microg per decade; P<.02). Independently of age, the amount of GH secretion was significantly associated with slow wave sleep (P<.001). Increasing age was associated with an elevation of evening cortisol levels (+19. 3 nmol/L per decade; P<.001) that became significant only after age 50 years, when sleep became more fragmented and REM sleep declined. A trend for an association between lower amounts of REM sleep and higher evening cortisol concentrations independent of age was detected (P<.10). CONCLUSIONS: In men, age-related changes in slow wave sleep and REM sleep occur with markedly different chronologies and are each associated with specific hormonal alterations. Future studies should evaluate whether strategies to enhance sleep quality may have beneficial hormonal effects. JAMA. 2000;284:861-868

Pathophysiology of human circadian rhythms.

The 24 h profiles of hormonal secretions represent a good model for the study of the human circadian system. Diurnal hormonal variations generally reflect the modulation of ultradian or pulsatile release at 1-2 h intervals by signals occurring at nearly 24 h periods and result from the interaction of an internal timekeeping system--or circadian clock--with the sleep-wake homeostasis and various environmental factors, including the light-dark cycle, periodic changes in activity levels and the meal schedule. This temporal organization is altered in many pathophysiological conditions, including ageing, sleep loss, night or shift work, jet lag, affective disorders and endocrine diseases. Both photic and non-photic stimuli may affect the regulation of the circadian pacemaker and, therefore, the diurnal pattern of hormonal secretions. Appropriately timed stimuli may induce either a phase-advance or a phase-delay of the circadian clock, according to the timing of administration. Phase-shifting effects have been shown in humans for light and for dark pulses, physical exercise, melatonin and melatonin agonists, and benzodiazepine hypnotics. These results open new perspectives for the treatment of a variety of disorders involving dysregulation of the circadian rhythmicity.

Daytime naps in darkness phase shift the human circadian rhythms of melatonin and thyrotropin secretion.

To systematically determine the effects of daytime exposure to sleep in darkness on human circadian phase, four groups of subjects participated in 4-day studies involving either no nap (control), a morning nap (0900-1500), an afternoon nap (1400-2000), or an evening nap (1900-0100) in darkness. Except during the scheduled sleep/dark periods, subjects remained awake under constant conditions, i.e., constant dim light exposure (36 lx), recumbence, and caloric intake. Blood samples were collected at 20-min intervals for 64 h to determine the onsets of nocturnal melatonin and thyrotropin secretion as markers of circadian phase before and after stimulus exposure. Sleep was polygraphically recorded. Exposure to sleep and darkness in the morning resulted in phase delays, whereas exposure in the evening resulted in phase advances relative to controls. Afternoon naps did not change circadian phase. These findings indicate that human circadian phase is dependent on the timing of darkness and/or sleep exposure and that strategies to treat circadian misalignment should consider not only the timing and intensity of light, but also the timing of darkness and/or sleep.

Impact of sleep debt on metabolic and endocrine function.

BACKGROUND: Chronic sleep debt is becoming increasingly common and affects millions of people in more-developed countries. Sleep debt is currently believed to have no adverse effect on health. We investigated the effect of sleep debt on metabolic and endocrine functions. METHODS: We assessed carbohydrate metabolism, thyrotropic function, activity of the hypothalamo-pituitary-adrenal axis, and sympathovagal balance in 11 young men after time in bed had been restricted to 4 h per night for 6 nights. We compared the sleep-debt condition with measurements taken at the end of a sleep-recovery period when participants were allowed 12 h in bed per night for 6 nights. FINDINGS: Glucose tolerance was lower in the sleep-debt condition than in the fully rested condition (p<0.02), as were thyrotropin concentrations (p<0.01). Evening cortisol concentrations were raised (p=0.0001) and activity of the sympathetic nervous system was increased in the sleep-debt condition (p<0.02). INTERPRETATION: Sleep debt has a harmful impact on carbohydrate metabolism and endocrine function. The effects are similar to those seen in normal ageing and, therefore, sleep debt may increase the severity of age-related chronic disorders.

Metabolic effects of short-term elevations of plasma cortisol are more pronounced in the evening than in the morning.

To determine whether elevations of cortisol levels have more pronounced effects on glucose levels and insulin secretion in the evening (at the trough of the daily rhythm) or in the morning (at the peak of the rhythm), nine normal men each participated in four studies performed in random order. In all studies, endogenous cortisol levels were suppressed by metyrapone administration, and caloric intake was exclusively under the form of a constant glucose infusion. The daily cortisol elevation was restored by administration of hydrocortisone (or placebo) either at 0500 h or at 1700 h. In each study, plasma levels of glucose, insulin, C-peptide, and cortisol were measured at 20-min intervals for 32 h. The initial effect of the hydrocortisone-induced cortisol pulse was a short-term inhibition of insulin secretion without concomitant glucose changes and was similar in the evening and in the morning. At both times of day, starting 4-6 h after hydrocortisone ingestion, glucose levels increased and remained higher than under placebo for at least 12 h. This delayed hyperglycemic effect was minimal in the morning but much more pronounced in the evening, when it was associated with robust increases in serum insulin and insulin secretion and with a 30% decrease in insulin clearance. Thus, elevations of evening cortisol levels could contribute to alterations in glucose tolerance, insulin sensitivity, and insulin secretion.

[Biologic rhythms. Effect of aging on the desynchronization of endogenous rhythmicity and environmental conditions]. / Les rythmes biologiques. Effets du veillissement et de la désynchronisation entre la rythmicité endogène et les conditions d'environnement.

CIRCADIAN AND PULSATILE RHYTHMICITY IN THE AGING PROCESS: The aging process produces morphological and neurochemical alterations in the suprachiasmatic nuclei as well as major alterations in the quality of sleep. In addition, aging is frequently accompanied by changes in life style due to different, often less demanding, social and occupational activities, leading to an attenuation of the synchronizing effects of the light-dark and activity-rest cycles. Together, these different elements contribute to a decline in temporal organization in the elderly, a phenomenon which starts in the third decade for some variables. There is a characteristic phase shift with age: in an 80-year-old individual, the circadian cortisol peak occurs about 3 hours earlier than in a 20 year-old-individual. JET LAG AND NIGHT SHIFT WORK: The circadian rhythm and environmental conditions can become desynchronized after transmeridian flights, a phenomenon commonly called jet lag. In night shift workers, such desynchronization creates an important public health problem. The impact may be underestimated since 15 to 20% of the work force in industrialized countries work permanently or occasionally on night shifts. The resulting dissociation between environmental signals and the wake-sleep cycle leads to various health problems. No truly effective therapeutic strategy has been developed although ongoing research, particularly on the use of light and/or melatonin, provides some promising perspectives.

[Biologic rhythms. Circadian, ultradian and seasonal rhythms]. / Les rythmes biologiques. Rythmes circadiens, ultradiens et saisonniers.

CIRCADIAN RHYTHMS: Our knowledge of the genetic and molecular mechanisms regulating the principal circadian clock located in the suprachiasmatic nuclei is progressing. The clock's intrinsic period varies from one species to another and to a lesser degree from one individual to another. In humans, the intrinsic period is slightly over 24 hours. The clock is capable of synchronizing itself to the surrounding environment by reacting to outside factors or zeitgebers (time-givers). Light-dark cycles are the main zeitgebers; meals, the social environment, and locomotor activity also affect the circadian clock. In addition, the circadian clock acts as an internal timer, providing the organism with a means of synchronizing the function of multiple biochemical and physiological systems. ULTRADIAN RHYTHMS: The frequency of ultradian rhythms varies considerably form one species to another and from one parameter to another. In humans, several functions oscillate at 60-120 minute intervals, rhythms which are sometimes superimposed on other functions oscillating at 3 to 5 minute intervals. SEASONAL RHYTHMS: Several mechanisms allow living organisms to adapt to seasonal variations in the environment. In certain species, reproduction functions are stimulated at appropriate moments in the yearly cycle, optimizing the newborn's chances of survival. Such seasonal variations are much less marked in humans.

[Biologic rhythms. Nyctemeral variation in man]. / Les rythmes biologiques. Les variations au cours du nycthémère chez l'homme.

CORTICOTROPIC AXIS: The nycthemeral pattern of cortisol is a good marker of the circadian clock. Cortisol levels fluctuate between a peak level, observed in the early hours of the morning, and a minimal level around midnight. This variability is considerably reduced or even abolished in Cushing s syndrome. THYREOTROPIC AXIS: The nycthemeral pattern of TSH secretion is dependent on both the circadian clock and sleep (which inhibits hormone secretion). The moment of the evening rise is a reliable marker of the circadian rhythmicity. SOMATOTROPIC AXIS: Growth hormone is essentially pulsatile. GH levels are often undetectable between pulses. The circadian rhythmicity plays only a minor role in the regulation of growth hormone secretion. LACTOTROPIC AXIS: Nycthemeral variations in prolactin secretion are mainly regulated by wake-sleep cycles; peak levels occur in the middle of the night. Prolactin secretion is also modulated by the circadian rhythmicity. GONADOTROPIC AXIS: Gonadotropins are secreted in pulses, following the pulses of GnRH secretion. In adult women, nycthemeral variations in LH are strongly modulated by the menstrual cycle. MELATONIN: The nychtemeral pattern of melatonin is an excellent marker of the circadian clock. Diurnal concentrations are low and vary little whereas peak levels are observed in the middle of the night. Melatonin rhythmicity is not influenced by sleep, but is dependent on exposure to light and darkness.