All life on earth has evolved in conditions of a 24-hour day-night (light-dark) cycle. Organisms have evolved mechanisms to time their cellular and metabolic processes to anticipate this daily rhythm. As a result, within nearly all cells of the human body there is a biological clock based on a cycle of DNA and protein synthesis. Clock gene activity has been found within white blood cells and cells of the heart, brain, liver and many other tissues.

The individual cellular clocks run on a cycle that is close to 24 hours. This is known as a circadian rhythm (“circa-” = about and “dian” = pertaining to a day). But because the clocks are not exact, the clocks of individual cells can drift apart from each other or from the earth’s day-night cycle. To keep these clocks in time there is a master clock located in the brain. In the same way that the conductor of an orchestra keeps the musicians playing in time with each other, this master clock keeps the body’s cellular clocks to the same time cycle.

The master clock is located in what is called the suprachiasmatic nucleus (SCN), located in a part of the brain called the hypothalamus which regulates many basic body functions. The SCN is composed of about 20,000 closely networked cells whose rhythms are coordinated so that the firing rate of the cells varies together in a near-24-hour rhythm. The firing of SCN cells is then transmitted directly and indirectly to many other regions of the brain which then pass on this clock signal to the rest of the body by neurochemical and hormonal means.

Two of the best characterized rhythms driven by the clock signal are the body temperature cycle and the production of the hormone melatonin. The SCN regulates body temperature via connections to other areas of the hypothalamus. Body temperature varies in a wave-like pattern, which reaches a maximum during the day and a minimum (or nadir) during the night.

The SCN also sends a nerve signal that follows a complex poly-synaptic pathway via the cervical spinal ganglia to regulate the activity of the pineal gland, which is responsible for the production of melatonin. Melatonin, sometimes called “the hormone of darkness,” is produced during the dark at night. It is secreted by the pineal into the cerebrospinal fluid and then travels in the bloodstream to reach the cells of the body. It acts upon specific melatonin receptors to directly regulate cell functions. It also reinforces the temperature cycle by facilitating the nocturnal drop in body temperature. Among other effects, this drop in body temperature helps ready the brain and body for sleep.

While the SCN serves to coordinate the cell clocks throughout the body, there is still a need to coordinate the SCN clock to the earth’s 24-hour period. If left to itself, the SCN keeps a rhythm that is close to but not exactly 24 hours. In healthy humans the intrinsic period of the SCN clock averages about 24.2 hours. If there were no way to correct this cycle to equal 24 hours the clock in the SCN would drift by several minutes each day until it no longer kept correct time or stayed “entrained.”

The primary means to keep the SCN clock set properly is via light-dark exposure. Specialized cells in the retina of the eye, which are different from the cells used for vision, register the exposure to light and dark and transmit this signal by a nerve path known as the retinohypothalamic tract to the SCN. When the eyes are exposed to light in the early morning hours this sends a signal that advances the clock in the SCN to an earlier time, thereby providing the necessary daily entrainment. When light falls on the eyes late at night, a delay signal is sent to the SCN. A graph of the effect of light at different times of day and night is known as a phase-response curve and can be used to predict the effects of light on the biological clock. If the SCN clock runs longer than 24 hours, it tends to become delayed relative to the day-night cycle, but morning light exposure will reset it. If the SCN clock runs shorter than 24 hours, late night light exposure will delay it a bit. By this means, the SCN clock is kept in time with the light and dark cycle of day and night. In healthy individuals routine exposure to morning light works to keep circadian rhythms entrained.

The retinal cells that register light for circadian functions use a pigment known as melanopsin as a light sensor. Because melanopsin is particularly sensitive to blue light, light of that color has a greater effect in circadian rhythms. Red, orange and yellow light have much less effect. Green light can also affect rhythms under certain circumstances.

Among the most important of the body rhythms controlled by the SCN is that of the sleep-wake cycle. This cycle is controlled by two processes known as the homeostatic process and the circadian process. During sleep the brain and body repair themselves and accumulate energy and metabolic resources for the activities of the day. During the day, while the person is awake, these resources are gradually consumed. The gradual loss of energy during the day produces a drive to sleep in order to restore that energy. This is known as the homeostatic sleep drive. If the homeostatic process were the only one involved, a person would wake up fully energized and then gradually wind down over the course of the day, like a battery losing power. This would mean an uneven level of alertness during the day, with dangerously low alertness in the afternoon and evening. To counterbalance this, the SCN also regulates alertness by what is known as the circadian process. As the day goes on, and energy winds down, the SCN compensates for this by sending a stronger alertness signal to the brain and body. This alertness signal reaches a peak in the two hours just before bedtime. This zone of maximum alertness is known as the “forbidden zone for sleep” since the alertness signal makes sleep nearly impossible during that zone. When the usual bedtime is reached, the SCN begins to turn down its alertness signal to allow the body to sleep. In order to prevent early awakening, before the night’s sleep is done, the circadian alertness signal declines further across the night.

This complex interplay between the circadian process and the homeostatic process allows the human organism to have a relatively level state of alertness during the day (with the occasional exception of a mid-afternoon nap period) and allows a 7-9 hour period of consolidated sleep at night.

When all is working well, light signals registered in the eyes keep the SCN on track with the 24-hour day-night cycle and the SCN in turn coordinates the clocks in the pineal gland and in cells throughout the body. All the clocks keep a 24-hour cycle in sync with each other like the members of a well-conducted orchestra. The circadian alertness signal then combines with the homeostatic process resulting in an individual who can sleep through the night and maintain alertness during the day.

But there are a number of things that can go wrong with this system and result in a circadian disorder such as N24.

1. Blindness. The most well-understood cause of N24 is what occurs in blind individuals. Persons who are completely blind (no perception of light) will not register the light signals which are needed to fine-tune the body clock to a 24-hour day. If the SCN clock starts to drift away from 24 hours, a blind person has no intrinsic way to bring it back in sync without medical treatment. Since the inherent rhythm of the SCN is not always precisely 24 hours, a blind person’s circadian timing system will slowly drift over time. They will cycle over time between periods of nighttime sleep and periods of daytime sleep. In the vast majority of cases the sleep rhythm gradually delays so the period is over 24 hours, but there are a few cases of gradual advances and a less-than-24-hour period. The length of the circadian period in blind persons with N24 is typically in the range of 23.8 to 25 hours.

2. Alterations in Light Sensitivity. In some sighted individuals there may be a subsensitivity or insensitivity to the effects of light on the circadian system. The vision-producing areas of the eye and brain may function well, but the separate cell pathway that transmits the circadian light signal may not. If they are totally insensitive to the circadian effects of light, their condition, from a circadian point of view, is not different from that of a blind person. If they are subsensitive to light, light may produce some effect on their rhythms but it may not be strong enough to correct circadian drift in their particular lighting environment.

Conversely, some patients with delayed sleep phase disorder, a condition related to N24, have been shown to be supersensitive to the effects of light. If they are exposed to normal room light in the evening it may produce an exaggerated delay in their circadian rhythms. If this delay becomes cumulative, the result is N24.

3. Environment. Environmental exposure to light may also play a role. Healthy individuals, when kept in isolation without time cues and allowed to turn their lights on and off when they choose, will often fall into a non-24-hour rhythm. The length of the rhythm is not only longer than the intrinsic 24.2-hour cycle of the SCN, but may be up to 25 hours or more in length. This is because self-selected light exposure late in the day has a delaying effect. However, this cannot be the sole cause of N24 since light does not lead to N24 in all persons in a non-isolated environment. In contrast, persons with N24 cannot maintain a 24-hour schedule even in a non-isolated environment with normal time cues.

4. Hormonal Factors. In some cases the hormone melatonin may be involved in the development or perpetuation of N24. Some patients with N24 produce less melatonin than normal, which can be problematic since melatonin helps link sleep to the day-night cycle. On the other hand, too much melatonin may also cause problems. The antidepressant fluvoxamine, which greatly increases melatonin levels by inhibiting its metabolism, has been reported to cause DSPD, which is closely related to N24. Some individuals have an abnormality in their ability to metabolize melatonin, which can lead to higher-than-normal daytime levels that may result in circadian clock dysfunction.

5. Differences in Cellular Clock Function. Other studies of the causes of circadian rhythm disorders have focused on the cellular clock itself. Studies in healthy subjects show a correlation between the period of the cellular clock and the phase of entrainment. Morning persons have a shorter clock period than evening persons. N24 may be an extension of extreme “eveningness” in which the cellular rhythm may be too far from 24 hours for normal light exposure to correct it, a situation known as being “outside the range of entrainment”.

The period of the human biological clock can be measured in two ways. First one may examine the period under the usual living conditions of the subject. Under those conditions the period of a normal person is 24 hours. The timing of their sleep wake cycle does not change over time. A person with N24 by definition will have a period that is longer than 24 hours, sometimes as long as 25-26 hours.

Under normal circumstances the circadian clock is affected by outside factors, especially light. But under special experimental conditions (constant routines and forced desynchrony) scientists can cancel out these outside effects and find what is called the intrinsic period of the clock. This is the time the clock would keep if it were isolated from outside influences. For normal subjects the intrinsic period of the clock is around 24.2 hours. Daily exposure to normal light compensates for the 0.2 difference and allows normal subjects to stay on a 24-hour day.

Three small studies have looked at the intrinsic period of N24 patients. One study of 6 patients found a 24.5 hour period, a study of 4 patients reported 24.9 hours, and a case report of a single patient also found a 24.5 hour period. Thus these N24 patients require an adjustment of at least 0.5 to 0.9 hours per day to remain in a 24-hour cycle. Normal light exposure may not be enough to make this adjustment. When combined with other factors that push the clock later this may make entrainment to a 24-hour day impossible.

Other studies have also looked at the clock within muscle cells (fibroblasts) extracted and grown in culture. The period of cells in culture is correlated with the intrinsic period of the person from whom the cells were sampled. This shows that the clock period is determined on a cellular level. For N24 patients this suggests that at least some may be manifesting a fundamental malfunction of the biochemical basis of the circadian clock, which results in a longer intrinsic period.

While the intrinsic period of N24 patients is longer than average, it overlaps with the period found in a few extreme evening type subjects who do not have clinical N24. Thus, while the long intrinsic period is clearly a major contributing factor to the development of N24, there may also be additional factors involved, which make the difference between an extreme evening chronotype and free-running N24.

6. Differences in the Regulation of Sleep. Another possible set of causes of N24 is related to the homeostatic and circadian regulation of sleep. On average patients with N24 have a slightly greater sleep requirement than normal. In some cases this can be extreme. While a healthy person may sleep 8 hours and be awake for 16 hours, if someone needs 12 hours of sleep and then is awake for a normal 16 hours, their day will last 28 hours total. The change in the sleep cycle will in turn change the timing of light exposure, perpetuating an N24 cycle. Similarly, if someone is deficient in the homeostatic drive for sleep they may sleep a normal 8 hours but require 20 hours of awake time before sufficient homeostatic pressure accumulates to permit sleep, again resulting in a 28 hour day.

The timing of sleep in relation to internal circadian rhythms, also known as the phase angle between sleep and circadian rhythms, is abnormal in many cases of N24. Here phase angle is described in terms of the relationship between sleep timing and the circadian rhythm of body temperature. In healthy individuals the body temperature starts to drop shortly before sleep onset and reaches a minimum late in the sleep period — usually about 2 hours before waking. Persons with N24 tend to fall asleep very late relative to their temperature cycle and so the time between the temperature minimum and time of waking (sleep offset) may be 4 hours or more, even up to 8 hours in extreme cases. Since the body’s response to light-dark exposure is synched with the internal rhythms (such as core temperature) rather than the sleep-cycle per se, N24s with an abnormal relationship between sleep and circadian rhythms will sleep through the phase advance portion of their clock and not get the light they need on a daily basis to reset their clock. At the same time since they are awake late relative to their temperature cycle, they are exposed to light during the phase delay portion of the phase response curve. This tends to push their circadian rhythm in the direction of a much longer than normal day. This amplifies the effect of the already prolonged intrinsic period of N24 patients.

The circadian regulation of sleepiness is also important. Even healthy individuals have a “forbidden zone for sleep” that occurs an hour or two before normal bedtime and is associated with the maximum circadian alertness signal. In persons with N24 this forbidden zone occurs too late in the day and is too strong to permit sleep on a 24-hour cycle.

This pattern may be reinforced by certain effects of sleep and wake on alertness. When individuals wake after a prolonged period of sleep, they are often in a state of reduced alertness known as sleep inertia. In people with N24 this state of sluggishness and grogginess may be very powerful and persist for many hours. The longer they are awake the more alert they become. (This may be explained by an observation that brain cell circuits become more excitable with longer time awake.) When it comes time for them to sleep (if they are trying to stay on a 24-hour cycle) their alertness will have reached a high point and their heightened state of energy, even if brief, will not permit them to fall asleep at a normal time. In addition, patients with N24 may not want to try to fall asleep at this time because they finally feel awake, alert and productive.

7. Development. Development of the brain, and in particular the circadian and sleep centers, is another factor. In pervasive developmental disorders such as autism a relatively high frequency of occurrence of N24 and other circadian rhythm and sleep disorders has been noted. It is assumed that the circadian and sleep centers of the brain did not properly develop or are affected by other neurochemical or anatomical deficits. It may be that other N24s who do not have pervasive developmental disorders may have impaired development limited to the sleep and circadian brain centers.

8. Trauma. Physical damage to the brain, such as occurs from head injury has been noted to lead to N24 in previously healthy individuals. It is assumed that the head injury damages the sleep and circadian centers of the brain such as the hypothalamus or pineal gland. Similarly, brain tumors have been noted to lead to the development of N24. Circadian sleep disorders have been noted in survivors of tumors affecting the pons and the hypothalamus. Craniopharyngiomas are particularly likely to lead to sleep disorders. In some cases the damage is due to the tumor itself and in other cases to the effects of radiation treatment to the head. In one case an aneurysm near the SCN resulted in transient N24. There has also been a report of N24 following chemotherapy for Hodgkin’s lymphoma.

Under the heading of physical abnormalities, any factor that leads to total blindness, whether via genes, disease or injury, can lead to secondary N24.

9. Iatrogenic. N24 can also arise from attempts at treatment of the more common disorder, delayed sleep phase disorder (DSPD). One of the widely used treatments for DSPD is chronotherapy, in which the patient is instructed to gradually delay their bedtime and wake time up to three hours a day until they go around the clock to a more socially acceptable sleep-wake schedule. In essence this means temporarily adopting an N24 schedule. Unfortunately, in some patients, once an N24 schedule has been established it becomes nearly impossible to break. They have exchanged one circadian rhythm disorder, DSPD, for an even more disabling one, N24. There are several reasons why the N24 pattern is hard to break out of once established. One involves the timing of sleep relative to the temperature rhythm mentioned above. The other involves what is called the plasticity of the circadian system. That means that once an organism has been placed on a particular cycle, including a non-24-hour cycle, the circadian clock remembers that cycle and tries to continue it. The risk of N24 after chronotherapy has been known since the 1990s but many doctors continue to be unaware of the risk when recommending chronotherapy.

10. Genetics. There is increasing evidence of a genetic component to N24. In most cases it is not a simple inherited genetic condition (Medelian inheritance). Most patients with N24 do not have parents or close relatives with the condition. However, there do seem to be several genetic factors which can predispose someone to the development of N24.

One study found specific genetic changes (single nucleotide polymorphisms, SNPs) in the gene BHLHE40 in 4 patients with N24. As this gene encodes components of the cellular clock, such mutations may affect clock function leading to the abnormalities noted in N24.

A separate study of 67 N24 patients found an association with polymorphisms in the PER3 gene. PER3 also encodes a crucial component of the circadian clock. The same polymorphisms were associated with extreme evening chronotype – a genetic predisposition to functioning better late in the day, a tendency which is also noted in Non-24s. Variations in the PER3 gene (both SNPs and repeat numbers) are believed to affect free-running period (in animals), the homeostatic drive for sleep (in humans) and the response to light (in humans). All of these factors have been hypothesized, with some evidence, to be abnormal in N24.

DSPD, a condition related to N24, has been linked to the presence of a mutation in the CRY1 gene, which plays a role in the circadian clock, in a study of one family.

Several genome-wide association studies – genetic screenings of over 100,000 persons — have shown genetic associations with human chronotypes. While these studies did not involve N24 patients specifically, N24 is closely related to extreme evening chronotype, suggesting some of the same genetic factors may be relevant.

Taken together, both the specific studies of genes in Non-24 and the more general genetic studies of circadian rhythms strongly suggest that some individuals may have a genetic predisposition to the development of N24.