Fukushige et al. Journal of Physiological Anthropology 2014, 33:33
http://www.jphysiolanthropol.com/content/33/1/33

ORIGINAL ARTICLE

Open Access

Effects of tryptophan-rich breakfast and light
exposure during the daytime on melatoninsecretion at night
Haruna Fukushige1, Yumi Fukuda2, Mizuho Tanaka1, Kaoru Inami3, Kai Wada4, Yuki Tsumura5, Masayuki Kondo6,
Tetsuo Harada4, Tomoko Wakamura1 and Takeshi Morita2*

Abstract
Background: The purpose of the present study is to investigate effects of tryptophan intake and light exposure
on melatonin secretion and sleep by modifying tryptophan ingestion at breakfast and light exposure during thedaytime, and measuring sleep quality (by using actigraphy and the OSA sleep inventory) and melatonin secretionat night.
Methods: Thirty three male University students (mean ± SD age: 22 ± 3.1 years) completed the experiments
lasting 5 days and 4 nights. The subjects were randomly divided into four groups: Poor*Dim (n = 10),
meaning a tryptophan-poor breakfast (55 mg/meal) in the morning and dim light environment (<50 lx)
during the daytime; Rich*Dim (n = 7), tryptophan-rich breakfast (476 mg/meal) and dim light environment;
Poor*Bright (n = 9), tryptophan-poor breakfast and bright light environment (>5,000 lx); and Rich*Bright(n = 7), tryptophan-rich breakfast and bright light.
Results: Saliva melatonin concentrations on the fourth day were significantly lower than on the first day in the
Poor*Dim group, whereas they were higher on the fourth day in the Rich*Bright group. Creatinine-adjusted melatoninin urine showed the same direction as saliva melatonin concentrations. These results indicate that the combination ofa tryptophan-rich breakfast and bright light exposure during the daytime could promote melatonin secretion at night;
further, the observations that the Rich*Bright group had higher melatonin concentrations than the Rich*Dim group,
despite no significant differences being observed between the Poor*Dim and Rich*Dim groups nor the Poor*Brightand Rich*Bright groups, suggest that bright light exposure in the daytime is an important contributor to raised
melatonin levels in the evening.
Conclusions: This study is the first to report the quantitative effects of changed tryptophan intake at
breakfast combined with daytime light exposure on melatonin secretion and sleep quality. Evening saliva
melatonin secretion changed significantly and indicated that a tryptophan-rich breakfast and bright light
exposure during the daytime promoted melatonin secretion at this time.
Keywords: Bright light exposure, Circadian rhythm, Melatonin, Sleep, Tryptophan* Correspondence: morita@fwu.ac.jp
2
Department of Environmental Science, Fukuoka Women’s University, 1-1-1,
Kasumigaoka, Higashi-ku, Fukuoka 813-8529, Japan
Full list of author information is available at the end of the article© 2014 Fukushige et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in thisarticle, unless otherwise stated.

Fukushige et al. Journal of Physiological Anthropology 2014, 33:33
http://www.jphysiolanthropol.com/content/33/1/33

Background
Melatonin is a hormone secreted by the pineal gland in
the brain. Its secretion is controlled by the suprachiasmatic nucleus in the hypothalamus, the central biologicalclock of circadian rhythms in humans. Daily exposure tolight resets the phase of the clock in the suprachiasmatic
nucleus [1]. Melatonin secretion usually shows a peak at
midnight, and it is often used as an indicator of sleep quality and phase advances or delays of the sleep-wake cycle[2,3]. In the clinical field, clinical doctors and researchers
use melatonin to improve sleep quality: to treat insomnia and depression and to reduce jet lag, and it isrecommended by some clinicians as a preventive agent forbreast cancer [4-9].
Tryptophan is one of the essential amino acids and iscontained in proteins sourced from milk, eggs, meat,grains, and beans. It can cross the blood–brain barrier
and is transformed into serotonin in the brain, subsequently converted to melatonin. The biosynthesis of
melatonin is rate-limited by the activity of arylalkylamine
N-acetyltransferase [10]. Zawilska et al. reported thatarylalkylamine N-acetyltransferase activity is affected bylight; its activity declines in the photoperiod (while melatonin secretion also decreases) and increases in the scotoperiod (when melatonin secretion also increases) [11].
Therefore, tryptophan intake and the timing of light
exposure must be considered together if the effect oftryptophan upon melatonin secretion and sleep quality
is to be maximized.
Hudson et al. [12] reported that using protein as asource of tryptophan before sleep improved its quality atnight by amounts that were comparable with the intakeof pharmaceutical-grade tryptophan. Markus et al. [13]examined the effects of tryptophan intake (from different
sources) at breakfast on the plasma tryptophan/large
neutral amino acids (TRP/LNAA) ratio and mood. The
results showed that intake of a tryptophan-rich source fromhydrolyzed protein had significantly greater effects on theplasma TRP/LNAA ratio and improvement of mood than
did pure tryptophan and alpha-lactalbumin whey protein.
In addition, Markus et al. [14] measured depressive moodand perceptual-motor and vigilance performance insubjects under stress (who had high or low chronic stressvulnerabilities) after they had ingested tryptophan-rich
egg protein hydrolysate in the morning. The results
revealed that egg protein hydrolysate improved thedepressive mood in all subjects and perceptual-motorand vigilance performance in those subjects who had
low chronic stress vulnerability. These experiments were
performed with the aim of examining the effects of tryptophan intake in the morning on serotonin synthesis, which
improves mood and mental activity. However, an important factor for this aspect of tryptophan function – lightintensity during the daytime – was not considered,Page 2 of 9and effects on sleep and melatonin secretion were notinvestigated.
Nakade et al. [15] reported the relationship between
tryptophan intake and sleep quality (in children aged 2to 6) in a survey of daily breakfast composition, morning
light exposure, and sleep. They found that tryptophan
intake at breakfast, coupled with morning light exposure,
was associated with higher melatonin secretion and easier
onset of sleep the following night. There is also evidence
that tryptophan intake in the morning and light exposure
at night affect the following sleep; Wada et al. [16] reported
that tryptophan-rich breakfasts and low-color temperaturelight sources at night increased saliva melatonin concentration in University soccer club members. Nevertheless,these investigations were carried out without regulation ofseveral aspects of the individuals’ lifestyle, including overall diet and daytime light exposure.
The purpose of the present study is to investigate effects of tryptophan intake and light exposure on sleep
and melatonin secretion, by modifying tryptophan ingestion at breakfast and light exposure during the daytime,and measuring sleep quality and melatonin secretion atnight.

Methods
Subjects and group setting

The experiment was performed during September 17th
to 21st, 2012 (lasting 5 days and 4 nights) on 40 male
University students, none of whom had any mental disorders (Cornell Medical Index), food allergies, or weretaking drugs. The data from 33 of them (mean ± SD age:
22 ± 3.1 years, height: 173 ± 5.5 cm, weight: 61.3 ± 6.7 kg,
BMI: 20.5 ± 2.3) were analyzed as three subjects withdrew and four subjects’ saliva samples were too small tobe analyzed. The subjects were randomly divided into
four groups: Poor*Dim (n = 10), tryptophan-poor breakfast (55 mg/meal) in the morning (between 07:30 h and
08:00 h) and dim light environment (<50 lx) during thedaytime (between 07:30 h and 18:00 h); Rich*Dim (n = 7),
tryptophan-rich breakfast (476 mg/meal) and dim lightenvironment; Poor*Bright (n = 9), tryptophan-poor
breakfast and bright light environment (>5,000 lx); andRich*Bright (n = 7), tryptophan-rich breakfast and bright
light environment. It is more convenient and practical for
healthy young people living a normal, busy lifestyle if thetryptophan in their diet is derived from common foodstuffs; therefore, the tryptophan-poor breakfast consisted
mainly of vegetables, such as salad and orange juice,whereas the tryptophan-rich breakfast was loaded withproteins from food sources such as salmon and natto (fermented soybeans; a Japanese traditional food) rather thanfrom nutritional supplements. The experimental conditions and subjects’ physical characteristics for the fourgroups are shown in Table 1. Subjects’ age, height, weight,

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Table 1 The four experimental groups
Group
Number of subjects

Poor*Dim

Rich*Dim

Poor*Bright

Rich*Bright

10

7

9

7

1

Age (years)*

22.1 (4.6)

21.4 (2.5)

21.0 (2.4)

21.4 (2.1)

Height (cm)*1

170.9 (6.1)

175.1 (5.1)

173.1 (4.0)

174.3 (6.3)

Weight (kg)*1

57.7 (5.6)

62.1 (7.4)

64.1 (7.1)

62.0 (5.8)

BMI*1

19.9 (2.3)

20.3 (2.9)

21.2 (1.9)

20.6 (2.5)

Tryptophan content of breakfast
Lighting condition during the daytime

2

3

Poor*

Rich*

4

4

Dim*

Dim*

2

Rich*35Bright*5

Poor*

Bright**1Mean (SD) is shown for age, height, weight, and BMI.
*2Tryptophan content of 55 mg.
*3Tryptophan content of 476 mg.
*4< 50 lx between 07:30 h and 18:00 h.
*5> 5,000 lx between 07:30 h and 18:00 h.

and BMI were not significantly different between the fourgroups (one-way ANOVA). Table 2 shows the estimatedintake of energy and basic nutrients, including tryptophan,at breakfast, lunch, and supper. The breakfast menus were
designed to differ in tryptophan content (TRP-Poor and
TRP-Rich breakfasts) but have minimal changes in othernutrients and energy. For each group, breakfast, lunch, andsupper were the same on all experiment days. The energy
and nutrient intakes of the controlled food were calculated
according to the Standard Tables of Food Composition in
Japan (5th Revised and Enlarged Edition). Lunch and supper were controlled with contents that were recommended

Table 2 Energy and nutrient intake at breakfast, lunch,and supper
Energy and nutrients
Energy*1(kcal)

Tryptophan (TRP) (mg)Protein(g)

Breakfast
TRP-Poor TRP-Rich
378

579

Lunch Supper
810

723

55

476

285

318

5.6

36.2

25.6

27.4

Lipid

(g)

7.7

17.5

23.1

16.4

Carbohydrate

(g)

71.7

65.2

121.6

113.9

Sodium

(mg)

813

1118

1742

941

Vitamin B6

(mg)

0.22

0.61

0.49

0.7

Isoleucin

(mg)

162

1649

1021

1084

Leucin

(mg)

311

2855

1826

1964

Phenylalanine

(mg)

200

1731

1028

1096

Tyrosine

(mg)

149

1388

774

898

Valine

(mg)

223

1936

1177

1298

LNAA*2

(mg)

1045

9559

5826

6340

TRP/LNAA(mol/mol)

0.036

0.034

0.033

0.034

*1All energy and nutrient intakes of the controlled food in the experimentwere calculated according to the Standard Tables of Food Composition in
Japan, and the coefficients for each food constituent were different from
Atwater’s coefficients.
*2LNAA: Large, neutral amino acids (isoleucin, leucin, phenylalanine, tyrosine,and valine).

as appropriate for healthy young males by the Ministry of
Health, Labour and Welfare of Japan.
The experimental protocol was conducted in accordance with the Declaration of Helsinki and conformed tointernational ethical standards, and was approved by the
Ethics Committee at Fukuoka Women’s University. Subjects gave prior written, informed consent and appropriatecompensation was given to them after they had completed
the experiment.
Experimental procedure

Figure 1 shows the experimental protocol. For one weekprior to the experiment, subjects were asked to eat avegetable-based breakfast (for tryptophan depletion) andto keep to a regular sleep-wake cycle (retiring at 00:00 hand rising at 07:00 h). Throughout this week, subjects’ adherence to these requirements was checked by objective
measurement of activity with an Actiwatch (Mini-Mitter
Co. Inc., Oregon, USA) and requiring the subjects to fill ina diary detailing sleep and meals eaten.
The subjects entered individual rooms at 13:00 h on
the first day and stayed under a dim light condition(<50 lx, 3,000 K). A curtain was closed and ceiling lights
were filtered for the condition. They continuously wore
the Actiwatch and their sleep (including sleep efficiency
and sleep latency) was monitored throughout the experiment. During days 2 to 4, food and drink intake
was regulated (see above), and subjects were required
to refrain from watching any video display monitors(cell phone, computer, or TV) in order to regulate
light exposure and sensitivity to light. They spent all
their time in the chair between 07:00–00:00 h except
for using a bathroom. All subjects were allowed toread a book or magazine and/or listen to music, but not
to take naps. Breakfast was served between 07:30 h and
08:00 h, lunch between 12:10 h and 12:40 h, and supper
between 19:10 h and 19:40 h. Subjects had 200 mL ofwater with each meal and were free to drink only water
between meals.

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Figure 1 Experimental protocol. Breakfast was served between 07:30 h and 08:00 h, lunch between 12:10 h and 12:40 h, and supper between
19:10 h and 19:40 h. For days 2 to 5, subjects rose at 07:00 h and answered the OSAsleep inventory after their urine sample had been collected(indicated by a white arrow underneath the time scale). Subjects in groups Poor*Dim and Rich*Dim continuously stayed under dim light (<50 lx)
whereas subjects in groups Poor*Bright and Rich*Bright stayed under bright light (>5,000 lx) between 7:30 h and 18:00 h. Saliva was collected
every hour between 18:00 h and 00:00 h (black arrows) and subjects retired at 00:00 h after emptying their bladder.

For days 2 to 5, subjects rose at 07:00 h and answered
the OSA sleep inventory [17] after a urine sample
had been collected. Subjects in groups Poor*Dim andRich*Dim remained under dim light between 07:30 h and
18:00 h whereas subjects in groups Poor*Bright andRich*Bright stayed under bright light (>5,000 lx) withnatural sunlight by opening curtain and artificial light byhaving a lighting device (Bright Light Me, Solartone Ltd.,Tokyo, Japan) placed in front of them. Under the bright
light condition, natural light changed color temperature accordingly. It was not compulsory for subjects to gaze at thelighting devise. Vertical illuminance and color temperature
at eye level were checked for dim and bright conditions
by using an illuminometer (Photo Recorder PHR-51,
T & D Co., Nagano, Japan) and an illuminance spectrophotometer (CL-500A, Konica Minolta, Inc., Tokyo,
Japan) respectively.
Saliva was collected into collection tubes with a purecotton swab (Sarstedt AG & Co., Tokyo, Japan) every
hour between 18:00 h and 00:00 h on days 1 to 4. Saliva
samples were immediately centrifuged at 1,500 g for
5 min after the collection, and the collection tubes without the cotton swab were then frozen at −30°C until
analysis. Subjects retired at 00:00 h after emptying their
bladder and their urine was collected between 00:00 h and
07:00 h. The urine samples were also stored at −30°C.
Subjects were free to leave after breakfast on day 5. All
urinary and salivary analyses were carried out in duplicate
and the mean values of the duplicates were used for statistical analyses.

Analysis method

The following factors were analyzed: i) sleep efficiency
and sleep latency from the actigraphs analyzed by the
Actiware-Sleep software, as measures of objective sleep
quality; ii) subjective estimates of sleep quality on rising,using the OSA sleep inventory (sleepiness, sleep maintenance, worries, integrated sleep feeling, and sleep initiation); and iii) melatonin concentrations in the risingurine sample and saliva samples taken in the 6 hours before retiring. Creatinine-adjusted melatonin, which was
not metabolized and was excreted in urine was represented as nighttime melatonin secretion in urine.
Urine samples were analyzed with RIA kits (RK-MEL2
200 tests, Bühlmann Laboratories AG, Schönenbuch,
Switzerland). The mean coefficients of variation (CV) of
the intra- and inter-assay precision were 7.9% and 11.7%,
respectively. The limit of detection (LoD) was 0.3 pg/mL
and the limit of quantification (LoQ) was 0.9 pg/mL. Regarding creatinine analysis, Creatinine kits (DeterminerL
CRE, Kyowa Medex Co. Ltd., Tokyo, Japan) were used.
The mean CVs of the intra- and inter-assay precisionwere <5% and <3%, respectively. The LoD was 0.04 mg/
dL and the LoQ was 0.1 mg/dL. Saliva samples were analyzed with RIA kits (RK-DSM2 200 tests, BÜHLMANN
Laboratories AG, Schönenbuch, Switzerland). The mean
CVs of the intra- and inter-assay precision were 7.9% and9.8%, respectively. The LoD was 0.2 pg/mL and the LoQ
was 0.9 pg/mL.
The dim light melatonin onset (DLMO) was determinedby linear interpolation between adjacent saliva samples

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using a fixed threshold of 3 pg/mL [18]. For five subjects,
their melatonin levels did not rise above the absolute
threshold (3 pg/mL) during the sampling period (18:00–
00:00 h). Therefore, their data were excluded from DLMO
analyses. Phase shift of saliva melatonin secretions wasconsidered by the difference of the DLMOs on days 1 and
4 (Day 1–Day 4) and statistically analyzed by using oneway ANOVA.
Two-way ANOVA for repeated measurements (Groupand Day, Time and Group, or Time and Day) was used
for sleep efficiency and sleep latency from the actigraphs,
the OSA sleep inventory, and saliva and urine melatoninconcentrations; in addition, for saliva melatonin concentrations, three-way ANOVA for repeated measurements(Group, Day, and Time) was also used. Differences in main
effects were investigated using Bonferroni corrections.
The analyses were performed useing SPSS (Ver. 19,
IBM, Tokyo, Japan), and a P value of <0.05 was considered to be statistically significant.

Results
There were no significant differences (between days 2
and 5 and the four groups) or interactions (between Groupand Day) with regard to sleep efficiency and sleep latency,as assessed from the actigraphs (two-way ANOVA, Groupand Day; Figure 2). However, even though there were no
significant differences between scores for the individual groups (multiple comparisons using the Bonferroni
method), ANOVA indicated that there were statisticallysignificant differences between the four groups overall
in the scores for sleepiness (P <0.01), sleep maintenance(P <0.05), and worries (P <0.05) from the OSA sleep
inventory (Group and Day; Figure 3). Main effects showedthat sleepiness and worries scores differed significantlywith Day (sleepiness, F (1, 33) = 6.0, P <0.05; worries,
F (1, 33) = 10.9, P <0.01) when they were compared on
the second and fifth days. The sleepiness score was 27.3 ±
1.0 (mean ± SE) on the second day and 29.3 ± 1.0 on thefifth day, and the worries score was 27.7 ± 0.9 on the second day and 30.2 ± 1.0 on the fifth day, which means that

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sleep quality was better (estimated as the result of sleepiness) and they were less worried on the fifth day compared to the second. Furthermore, although there were no
significant differences between the scores on days 2 and 5,
in Rich*Bright, the mean scores of sleepiness, sleep maintenance, worries, integrated sleep feeling, and sleep initiation on day 5 were higher than on day 2.
Regarding saliva melatonin concentration, there was asignificant interaction between Group and Day (F (3, 29) =6.2, P <0.01), but no interaction was found in relation to
Time (three-way ANOVA; Group, Day, and Time). Saliva
melatonin concentrations showed significant differences
according to the time of the evening (F (6, 174) = 49.6,
P <0.01).
In a further analysis of the effects of tryptophan andlight condition upon saliva melatonin, concentrations
during the evenings of days 1 and 4 were separately
assessed by ANOVA for differences between Group andTime (Figure 4). On day 1, there were no significant differences between the four groups whereas, on day 4,
the concentrations in the groups differed significantly(P <0.01). Multiple comparisons showed that, on day 4,
there were significant differences between Poor*Dim andRich*Bright (P <0.01) and Rich*Dim and Rich*Bright(P <0.01). Furthermore, saliva melatonin concentrations
during the evenings of days 1 and 4 were compared by
ANOVA for differences between Time and Day (Figure 5).
There were significant differences between the concentrations on days 1 and 4 in the groups Poor*Dim (F (6, 54) =5.0, P <0.05) and Rich*Bright (F (6, 36) = 2.9, P <0.05), but
not in Rich*Dim and Poor*Bright. Compared with the firstday, saliva melatonin secretion on the fourth day was
lower in Poor*Dim but higher in Rich*Bright.
On days 1 and 4, the DLMO was considered for phaseshifts of melatonin secretion. Table 3 shows the DLMOs of
the four groups. There were significant differences between
the shifts of DLMOs (Day 1–Day 4) in the four groups(one-way ANOVA, Group, P <0.01). Multiple comparisons showed that there were significant differences between Poor*Dim and Poor*Bright (P <0.01), Poor*Dim

Figure 2 Mean score (and SDs) for sleep efficiency (a) and latency (b) of the four groups on days 2 (white bars) and 5 (filled bars).

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Figure 3 Mean score (and SDs) for scores of the OSA sleep inventory in the four groups on days 2 (white bars) and 5 (filled bars).
(a) Sleepiness, (b) Sleep maintenance, (c) Worries, (d) Integrated sleep felling, and (e) Sleep initiation.

and Rich*Bright (P <0.01), Rich*Dim and Poor*Bright(P <0.01), and Rich*Dim and Rich*Bright (P <0.01). The
DLMOs were significantly advanced by bright light exposure but not by a tryptophan-rich breakfast.
Creatinine-adjusted melatonin in urine in the risingsamples taken on days 2 and 5 are shown in Figure 6.
In groups Poor*Dim and Rich*Dim, the mean valuesof the concentrations were lower on day 5, whereas,in Poor*Bright and Rich*Bright, they were higher on
day 5. These results showed the same direction as saliva
melatonin secretion. However, there were no statistically significant differences between the days, and no
significant interaction between the Group and Day(two-way ANOVA).

Figure 4 Average saliva melatonin concentrations during the evenings of day 1 and day 4.

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Figure 5 Average saliva melatonin concentrations (and SDs) during the evenings of days 1 and 4 in each group. Note that the data
show the same as those in Figure 4.

Discussion
The effects of a tryptophan-rich or tryptophan-poor
breakfast and bright or dim light exposure during daytime
upon sleep efficiency and latency (using actigraphy), fivesubjective aspects of sleep (from the OSA sleep inventory),and saliva and urine melatonins have been examined.
Although there were no significant group differences
between saliva melatonin concentrations on the first day,significant differences were present on the fourth (Figure 4).
The significant differences were between the Poor*Dim
and Rich*Bright groups and between Rich*Dim andRich*Bright groups (in both cases being higher in the
Rich*Bright group). These results indicate that a tryptophanrich breakfast and bright light exposure during daytime
could promote saliva melatonin secretion at night; further,
the observations that the Rich*Bright group had higher

melatonin concentrations than the Rich*Dim group despite no significant differences between Poor*Dim andRich*Dim nor Poor*Bright and Rich*Bright, suggests thatbright light exposure in the daytime is an important contributor to raised melatonin levels in the evening, as has
previously been found by others [19-22]. In addition, theDLMOs of saliva samples were significantly advanced bybright light exposure (Table 3). It is reported that bright

Table 3 DLMOs of the four groups on days 1 and 4
DLMO*1Group

Number of
subjects

Day 1

Day 4

Day 1–Day 4 (min)Poor*Dim

8

21:53 (54)22:12 (58)−19 (13)

Rich*Dim

6

21:30 (60)

22:36 (47)−66 (14)

Poor*Bright7

21:23 (64)

19:57 (75)86 (32)

Rich*Bright7

21:38 (60)

20:07 (52)90 (53)*1Mean time of day and SDs are shown for DLMO on days 1 and 4.

Figure 6 Mean creatinine-adjusted melatonin concentration(and SDs) of the four groups in the rising sample taken on days
2 (white bars) and 5 (filled bars).

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light exposure (5,000 lx) in daytime (11:00–17:00 h) raised
the following melatonin secretion at night and advanced
its phase shift [19]; the results herein were in agreementwith this report.
As shown in Figure 5, comparisons of saliva melatoninconcentrations on the first and fourth days showed significant differences in groups Poor*Dim and Rich*Brightbut no significant differences in groups Rich*Dim andPoor*Bright. The differences between days 1 and 4 werein the opposite direction in groups Poor*Dim andRich*Bright; in the Poor*Dim group, melatonin secretion on the fourth day was lower than on the first day,whereas it was higher on the fourth day in Rich*Bright. It
remains to be investigated if this change in the timecourse of melatonin secretion observed in the evening
continues until the following morning – that is, if there
are phase advances/delays in the secretory profile and/or if there are changes in the total amount of melatoninsecreted during the course of the whole night. However, in urine melatonin secretion, compared with thesecond day, the mean melatonin concentration on thefifth day was lower in Poor*Dim but higher in Rich*Bright(Figure 6). Although not statistically significant, these results show the same direction as found in saliva concentration (Figure 5). Therefore, the changes in melatoninsecretion in saliva could indicate an increase of melatoninsecretion and/or a phase shift of melatonin secretion. This
point will be investigated by measuring saliva melatoninconcentrations after midnight in a further study. Furthermore, only in groups Poor*Dim and Rich*Bright there
were significant differences between saliva melatoninconcentrations on days 1 and 4, and no significance wasfound in groups Rich*Dim and Poor*Bright (Figure 5).
These results indicate that a combined effect of tryptophan intake and bright light exposure – an increase of salivary melatonin after a tryptophan-rich breakfast wasaugmented by bright light exposure during the daytime
and vice versa – provides information that can be used topromote a healthy lifestyle.
Increased melatonin secretion raises the issue of changes
in sleep. Although no significant differences were foundfor objective measures of sleep efficiency and latency, subjective feelings of sleepiness and worries were significantly
improved on the fifth day compared to the second. In
addition, only in Rich*Bright, all mean scores (sleepiness,sleep maintenance, worries, integrated sleep feeling, andsleep initiation) on day 5 were higher than on day 2. Although there were no significant differences between thescores on days 2 and 5, it is possible that tryptophan intake and bright light exposure improve subjective sleep
quality. However, since there were no significant differences between the four groups, it is also possible that
there was a general improvement in sleep over the courseof the experiment, unrelated to the treatments. This

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improvement might result from the subjects becoming accustomed to the conditions in the experimental chamber.
Furthermore, effects of light and tryptophan might depend
upon the age and sensitivity of subjects [13,23]. Since thesubjects were young and healthy, the tryptophan and lighteffects might not be strong enough to cause clear sleepchanges, although our bright condition (>5,000 lx) was
bright enough to occur only rarely in most individuals’
daily lives. The generality of the present findings remains
to be elucidated; for example, whether these results apply
to females, older subjects, and non-Japanese populations
whose basic diet is different.
It should also be considered why a tryptophan-richbreakfast and daytime exposure to bright light had noeffects on objective (actigraphic) measures of sleep efficiency and latency. One reason might be due to limitations of the Actiwatches, which, although convenient forthe subject, do not assess sleep architecture, they overestimate sleep latency, total sleep time, and sleep efficiency,
and underestimate intermittent awakenings [24,25]. Another reason might be the sample size used in this study; alarger sample size would help increase the accuracy ofdata analysis and the small group sizes might have led tothe present negative results (a Type 2 error). In addition,
Silber and Schmitt [23] suggested that, in healthy adults,
tryptophan intake during the daytime had a relaxing andcalming effect whereas taking it at night might have only aminimal effect on the subsequent sleep [23]. Therefore,
the effects of the timing of tryptophan intake upon moodand sleep require further study.
The study design is also limited with regard to an absence of complete control of the food eaten at breakfast;
although the TRP/LNAA ratio was controlled, as shownin Table 2, other nutrients and calorie intake differed. It
is possible that these other factors affected the OSA
scores and melatonin secretion. A stricter regulation of
the subjects’ diet would be required to investigate theseeffects, even though complete control of the diet would
be difficult to achieve. One way to investigate an effectof tryptophan independent of other nutrients is to administer pure tryptophan without food manipulation, as
was done by Markus et al. [13,14]. Their results showedthat pure tryptophan and tryptophan-rich protein hydrolysate had greater effects on the plasma TRP/LNAA
ratios and brain tryptophan availability, and thus caused
mood and performance improvements. The effects ofpure tryptophan or tryptophan-rich protein hydrolysate
on melatonin secretion and sleep will be investigated in
our next study.

Conclusions
This study is the first to report quantitative effects ofchanged tryptophan intake at breakfast combined withdaytime light exposure on melatonin secretion and sleep

Fukushige et al. Journal of Physiological Anthropology 2014, 33:33
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quality. Evening saliva melatonin secretion changed significantly and indicated that a tryptophan-rich breakfast
and bright light exposure during the daytime promoted
melatonin secretion at this time.
Abbreviations
CV: Coefficients of variation; DLMO: Dim light melatonin onset; LoD: Limit ofdetection; LoQ: Limit of quantification; Poor*Bright: Tryptophan-poor
breakfast and bright light environment; Poor*Dim: Tryptophan-poor breakfast
and dim light environment; Rich*Bright: Tryptophan-rich breakfast and bright
light environment; Rich*Dim: Tryptophan-rich breakfast and dim lightenvironment; TRP/LNAA: Tryptophan/large neutral amino acids ratio.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
The authors alone are responsible for the content and writing of the paper.
All authors read and approved the final manuscript.
Author details
1
Department of Human Health Sciences, Graduate School of Medicine, Kyoto
University, 53, Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
2
Department of Environmental Science, Fukuoka Women’s University, 1-1-1,
Kasumigaoka, Higashi-ku, Fukuoka 813-8529, Japan. 3Department of Living
Environmental Science, Fukuoka Women’s University, 1-1-1, Kasumigaoka,
Higashi-ku, Fukuoka 813-8529, Japan. 4Laboratory of Environmental
Physiology, Graduate School of Integrated Arts and Sciences, Kochi
University, 2-5-1, Akebonocho, Kochi 780-8520, Japan. 5Department of Foodand Nutrition, Junshin Junior College, 1-1-1, Chikushigaoka, Minami-ku,
Fukuoka 815-8510, Japan. 6Comprehensive Housing R & D Institute, Sekisui
House Ltd, 6-6-4, Kabutodai, Kizugawa-city, Kyoto 619-0224, Japan.
Received: 21 May 2014 Accepted: 23 October 2014
Published: 19 November 2014
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doi:10.1186/1880-6805-33-33
Cite this article as: Fukushige et al.: Effects of tryptophan-rich breakfast
and light exposure during the daytime on melatonin secretion at night.
Journal of Physiological Anthropology 2014 33:33.

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