Effects of a Snack on Performance and Errors During a Simulated 16-h Night Shift: a Pilot Study

Background: Night shift workers might not eat due to their busy schedules during the night shift. However, diet may not only satisfy hunger, but also affect performance and errors. The aim of this study was to clarify the effect of a snack on performance and errors during 2-day, 16-h simulated night shifts. Methods: A randomized repeated-measure crossover study was performed to investigate subjective and cognitive performance in 15 healthy female adults (mean age, 21.7 years) after they consumed a snack (352 kcal) during a simulated night shift (16:00 to 09:00). The participants were kept awake from wake up in the morning to the next day at 09:00. Subjects were tested for performance on the Uchida-Kraepelin test, as well as for subjective feeling, body temperature, psychomotor vigilance test, and heart rate variability, before and after they consumed the snack. One day before the experiment, all participants wore an actigraphy monitoring device to determine their sleep state. Results: There was no difference between the snack condition and the skipping condition in sleep states the day before the experiment. On the day of the experiment, between 16:00 and 09:00, subjective sleepiness, fatigue, and body temperature were not different between the two conditions. Subjects showed a signicant improvement in performance on the Uchida-Kraepelin test and total errors on the psychomotor vigilance test, the primary outcome measure, when they consumed a snack compared with the skipping condition. In addition, the snack condition was associated with decreased high-frequency power, decreased low-frequency power/high-frequency power ratio, and increased heart rate, in the vagally mediated heart rate variability indices, which may reect a higher ability to modulate cognitive and behavioral processes. Conclusions: These results suggest that providing a snack to shift workers during night shifts might improve work safety and eciency.

addition, it has been reported that long working hours are associated with a higher risk of errors and accidents and poorer performance [5]. Compared with the traditional 8-h working day, the risk of occupational injury when working more than 12 h per day is estimated to be 147% higher [6].
Night work has been reported to increase morning sleepiness because of disruptions in circadian rhythms [7]. In addition, regarding dietary patterns during night shifts, avoiding eating altogether has been investigated as a strategy to maintain alertness [8]. A previous study [9] investigating the effects of food intake during nighttime (from 01:30 to 02: 30) found that fasting resulted in reduced performance in a cognitive vigilance task. Another study reported that eating a snack during the night shift (at 01: 30) alleviated hunger without leading to perceived fullness or increased sleepiness until 05:00 [10], although the snack's effect duration has remained unknown. Portion size is an important aspect of nighttime eating. Among nurses, reduced meal sizes have been reported as a strategy for minimizing gastric symptoms during night shifts [8]. However, to the best of our knowledge, the duration of the effects of food intake and the potential impact on performance have not been reported in previous studies investigating the effects of sleep loss on performance. Given this background and the fact that performance is most severely impaired at night, reducing food intake during night shifts may serve as a suitable countermeasure. If the duration of the snack effect could be clari ed, it could help answer the core questions of both what and when night shift workers should eat to avoid metabolic disturbances; this, in turn, could optimize wakefulness and performance and help prevent accidents in the workplace.
Accordingly, in this study, a rice ball was selected as a midnight snack that night shifters can easily eat even if they are busy. Rice is a staple food for more than half of the world's population. In addition to calories, rice is a good source of magnesium, phosphorus, manganese, selenium, iron, folic acid, thiamin, and niacin, but it is low in ber and fat.
Therefore, this study aimed to investigate the impact of eating (the Snack condition) compared with not eating (the Skipping condition) at night on performance, sleepiness, fatigue, and other physiological measures during 16-h simulated night shifts. We hypothesized that under the conditions of extended wakefulness during the "biological night," eating a snack would lead to decreased errors and maintain performance until the morning. To test this hypothesis, sleepiness, hunger, vigilant attention, cognitive processing, and other physiological measures were assessed under both Snack and Skipping conditions during consecutive 2-day, 16-h simulated shifts throughout the night.

Participants
The participants in this randomized, crossover study were 15 healthy adult females (mean age ± standard deviation [SD], 21.7 ± 0.5 years; body mass index, 19.81 ± 2.45 kg/m 2 ) recruited between August and September 2018. None of the subjects had any previous night shift experience, and none was identi ed as morning type or evening type according to the morningness-eveningness questionnaire [11]. All of the participants were current nonsmokers, in the luteal phase of their menstrual cycle [12,13], and had normal sleep patterns (habitual sleep ranging between 7 and 9 h). No participants were currently taking any prescribed medications. The required sample size was determined to be 14 (actual power 81.0%) based on an effect size, a error, and power (1-b) of 0.25, 0.05, and 0.8, respectively. The power calculation in this study was carried out using G*Power 3 [14].

Study meal
All participants consumed one or two meals between 16:00 and 09:00. Under both the Snack and Skipping conditions, all participants ate a meal (containing 708 kcal, 19.4 g protein, 17.9 g fat, 112.4 g carbohydrates, and 3.5 g sodium) at 19:30 (not eating at night), and under the Snack condition only, participants ate a snack at 03:30 (eating at night). The meal provided at 03:30 consisted of two rice balls (containing 352 kcal, 5.8 g protein, 1.8 g fat, 75.8 g carbohydrates, and 1 g sodium) and two slices of yellow pickled radish. Many night shift workers prefer a high-energy diet rich in carbohydrates [15], such as the rice balls that are widely available at any convenience store in Japan. All participants were given 20 min to nish their meals, and they were encouraged to eat everything on their plate.

Study protocol
A owchart of this study is shown in Figure 1. The measurements were conducted over 2 consecutive days between 16:00 and 09:00. Each experiment day had 5 participants who were randomly assigned to one of two conditions using counterbalancing. All participants were instructed to refrain from strenuous physical exercise and not to consume caffeine or alcohol for 24 h prior to and during each study day. Two days before the experiment began, all participants wore an actigraphy monitoring device (ActiGraph; Ambulatory Monitoring Inc., Ardsley, NY, USA) on their non-dominant wrist and recorded their sleep and activity levels in a diary.
On the day of the experiment, all participants arrived at the laboratory at 15:00. Until 16:00, they carried out practice assessments, including the Uchida-Kraepelin test (UKT) and the psychomotor vigilance test (PVT). The Snack condition involved consuming a meal and a snack at 19:30 and 03:30, whereas the Skipping condition involved consuming a meal at 19:30 only. At the start of each experiment, the participants were tted with a heart rate variability (HRV) sensor (GMS Inc., Tokyo, Japan). For each hour throughout the experiment, they were given 10 min to record their sublingual temperature once and complete the visual analog scale for sleepiness, fatigue, and hunger, 10 min to perform the UKT, and 10 min to measure the PVT. The next 20 min were considered free time, and the remaining 10 min were considered a rest period. The participants spent their free time reading, drawing, or drinking water. During the 10-min rest period, they sat on chairs and chatted with the other participants. The same meal amounts and contents were given to the participants between 19:30 and 19:50 in each experimental period. The HRV sensor was removed at the end of each experiment, but the participants were asked to continue wearing the actigraphy monitoring device until they woke up the next day. During all waking times, the participants remained awake in the laboratory and were continuously monitored by the researchers.
All participants stayed in a windowless and sound-insulated laboratory for 2 consecutive days (1 night) ( Figure 1). The laboratory environment was maintained at 26 ± 2 °C [16] and 50% relative humidity under indoor illumination on the table at 200 lx.

Sleep parameters
The sleep parameters measured were total sleep time, sleep e ciency (total sleep time / time in bed 1 00), sleep onset latency, and wake after sleep onset. All parameters were measured using the actigraphy monitoring device, and they were analyzed using the AW2 software package (Ambulatory Monitoring Inc.).

Sublingual temperature
The circadian rhythm of body temperature is one of the most frequently used indicators of circadian rhythmicity [17], and body temperature has been shown to be related to sleepiness, fatigue, and performance of a single-digit mental arithmetic task [18]. Sublingual temperature, which is considered an index of internal body temperature [19], was measured hourly using an oral thermometer (MC-612; Omron Inc., Kyoto, Japan) to assess changes in circadian modulation during the night.

Cognitive performance
Subjective assessment of sleepiness, fatigue, and hunger A visual analog scale was used for the subjective assessment of sleepiness, fatigue, and hunger [20]. The participants rated their sleepiness, fatigue, and hunger every hour on a 100-mm line, with values ranging from 0 mm (not sleepy, tired, or hungry at all) to 100 mm (extremely sleepy, tired, or hungry).

Uchida-Kraepelin Test (UKT)
The UKT (Nisseiken, Tokyo, Japan), a serial mental arithmetic task, was used to measure cognitive performance. This test is a questionnaire that requires intense concentration and effort, and it has been used as a tool to induce mental stress [21]. The test material consisted of a pre-printed paper with 20 rows of 115 random, single-digit gures. The subjects' task was to add adjacent gures horizontally, and then write the one-digit answer between the 2 gures; they were asked to proceed along each row as quickly and as accurately as they could in a 1-min period. On being given the rst cue, the subjects began calculating from the rst row. Then, when the second cue was given after 1 min, the subjects were required to begin a new row, without regard to their position on the current row. This procedure was repeated 8 more times, for the total duration of 10 min. The sum of the correct answers for each 1-min period over the 10-min task was used as the value for the analysis.

Psychomotor vigilance test (PVT)
The PVT is a reaction time task considered to be a sensitive measure for assessing the effects of sleep loss [22]. In this study, a precise computer-based version of the 10-min PVT was used to avoid problems of uncertainty with regard to the accuracy of the test platform timing [23]. All participants were instructed to look at a computer monitor and press a response button when a white circular edge appeared on the screen; pressing the response button stopped the counter and displayed the response time (in milliseconds) for a 1-s period. The PVT measures response times to visual stimuli randomly occurring at 2-to 10-s intervals over a 10-min period [23]. The outcome measures for the PVT include the median response time, number of lapses (response time > 500 ms), and total errors (incorrect responses), as well as mean response time.

Autonomic nervous system activity
For the purposes of the present study, HRV was obtained through autoregressive analysis of R-R intervals measured between 16:00 and 09:00. All data were analyzed o ine after analog-to-digital conversion of 250-Hz R-R waves. HRV was measured every 5 min during each hour and then averaged; these measurements were used to monitor autonomic nervous system activity throughout the night [24]. High-frequency (HF) and the low-frequency/high-frequency (LF/HF) are used as indicators of cardiac parasympathetic and cardiac sympathetic nervous activity, respectively [25,26]. The LF/HF power ratio indicates the balance between sympathetic and parasympathetic out ows [27].

Statistical analysis
All results are shown as mean ± SD or standard error of the mean. All sleep variables measured the day before the experiment were analyzed using the t-test.
To test the effects of consuming a snack on neurobehavioral and physiological outcomes during the early morning measurement periods, a fully saturated, linear mixed-effects analysis of variance was carried out [28], with a between-participant xed effect of condition and a within-participant xed effect of time (at 03:00 vs. from 04:00 to 09:00) and a random intercept. Within-condition comparisons were used to minimize the effect of individual differences. Multiple comparisons were assessed using the Bonferroni correction to evaluate patterns of change under the two conditions. As a secondary analysis, between-condition hourly comparisons from 16:00 to 09:00 were analyzed using the Mann-Whitney U test.
To assess the postprandial effect of the meal throughout the testing time, the net incremental area under the curve (niAUC), calculated from pre-(at 03:00) and postprandial time points, was tested. All statistical analyses were conducted using SPSS (version 22.0J; IBM, Tokyo, Japan). The hypothesis rejection level for all tests was set at p < .05, and a notable trend was set at p < .1.

Ethical considerations
This study was approved by the Center for Integrated Medical Research of Hiroshima University (study protocol ID No.: C-252). Written, informed consent was obtained from all participants before the rst examination. The study protocol conformed to the Declaration of Helsinki guidelines. This study was registered with the University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CRT registry ID: UMIN 000034345) after the enrollment of participants had begun. The authors con rm that all ongoing and related trials for this intervention have been registered.

Results
Sleep state before the experiment Total sleep time, sleep e ciency, sleep onset latency, go to bed time, and wake up time, assessed by the ActiGraph, were not different between phases (Table 1). Date are means (SD), N=15

Physiological parameters
Outcomes regarding the participants' physiological parameters, cognitive performance, and autonomic nervous system activity from 16:00 to 09:00 are shown in Figures 2 and 3. From 16:00 to 03:00, no signi cant main effect of their interaction was observed, and the analysis from 03:00 to 09:00 is shown in Table 2.

Cognitive performance
The interaction between condition and time of measure was not signi cant for sleepiness and fatigue, such that sleepiness and fatigue increased in the two conditions from 03:00 to 09:00; a similar trend was seen for across the night to early morning ( Figure 2B, C, left panel).
The interaction between eating condition and time of measure was signi cant for hunger (p < .001; Table  2). As can be seen in Figure 2D (left panel), hunger decreased in the Snack condition compared to the Skipping condition (p < .001) from 04:00 to 09:00.
The results of the UKT are shown in Figure 3A. Signi cant main effects of condition were observed for the number of correct answers, in which the number of correct answers performed from 04:00 to 09:00 was signi cantly better in the Snack condition than in the Skipping condition (p < .001).
PVT Results regarding the mean RT, lapse, and total errors on the PVT from 03:00 to 09:00 are shown in Figure   3B-D. On PVT, mean RT, and lapse, there were no signi cant main or interaction effects. On the other hand, total errors showed a signi cant main effect of condition (p = .025); total errors were signi cantly fewer under the Snack condition than under the Skipping condition ( Figure 3D).

Autonomic nervous system activity
Electrocardiogram data could not be obtained from one participant during the experiment; therefore, data were analyzed for 14 participants. Figure 4A shows the heart rate data. Signi cant main effects of condition (p < .001) and their interaction were observed (p = .029). Under the Snack condition, heart rate increased compared with the Skipping condition at 04:00 (p = .028), 05:00 (p = .046), 06:00 (p = .028), and 07:00 (p = .078).

Discussion
The present study investigated the effects of nighttime eating during night shifts on temperature, subjective sleepiness, fatigue, hunger, vigilant attention, processing speed, and autonomic nervous system activity during testing between 16:00 and 09:00. Increasing perceptions of hunger across night shifts have been reported by shift workers, who have described hunger as a factor that in uences their decision to eat during the night shift [8]. In the present study, consuming a snack (352 kcal) during the night reduced perceptions of hunger, maintained better performance, and increased heart rate for 3 h. To the best of our knowledge, this study was the rst to investigate each hour the effects of consuming a snack on objective nighttime sleepiness and fatigue, vigilant attention, information processing, and changes in autonomic nervous system activity over a simulated night shift in a controlled laboratory environment.
Regardless of the Snack condition, the greatest impairments in subjective sleepiness and fatigue, vigilant attention, and performance increased toward morning. This could explain the increases in circadian sleep pressure and decreased alertness throughout the night [29]. Consistent with our previous study [30], although sleepiness and fatigue increased across the simulated 16-h night shift, no differences were found between participants that had and had not eaten during the night [10,31]. Whereas the results after 04:00 showed increased subjective sleepiness and fatigue [32], this might correspond with the circadian low points between 02:00 and 06:00 [29]. Previous studies have suggested that snacking (10% 24-h estimated energy requirement) at 00:30 did not increase sleepiness at 02:30 and 05:00 [10], but eating at 01:30 is not a suitable mealtime, because sleepiness increased at 03:00. In the present study, sleepiness and fatigue did not differ, regardless of eating. It is speculated that rice balls are mainly carbohydrates, low in fat and relatively slow to digest and absorb, and their granular shape is presumed to require chewing; in addition to the effect on motor control, chewing increases the arousal level and alertness [33]. Thus, these factors may have prevented a signi cant increase in drowsiness [33]. Therefore, we concluded that the ingestion of the snack (352 kcal) did not change the subjects' sleepiness and fatigue. Performance on the PVT re ects circadian modulations in neurobehavioral functions, in addition to the effects of sleep pressure that develop with an increased duration of waking time, without being confounded by a learning curve [34]. PVT, mean RT, and lapse were similar between the two conditions. Subjective sleepiness is more likely to be underestimated [35] than objectively evaluated sleepiness [35]. Drowsiness weakens alertness and is likely to cause human error. In this study, the snack at 03:30 was affected by circadian modulations in neurobehavioral functions that developed in association with waking time and sleep pressure; decreases were seen in attention, but the PVT total errors were signi cantly higher in the Skipping condition than in the Snack condition. In particular, night shift workers are prone to errors at dawn, but it has been shown that light meals may make it possible to prevent accidents in the morning.
According to recent reviews, breakfast consumption may result in acute improvements of memory, attention, and motor and executive function, although no conclusion about the effect of macronutrients on cognitive function has been reached [33]. The present result shows that a small snack lessened the decline in the number of correct answers on the UKT. The UKT, which is used to measure cognitive task performance, involves simple mental arithmetic and handwriting [33]. Several cognitive domains, including sustained attention and short-term memory, are involved in the mental arithmetic task.
Handwriting is also a complex perceptual-motor skill [35]. Given recent studies highlighting the potential metabolic consequences of consuming a large nighttime meal [36,37], a small meal, such as two rice balls, may be an option that is more readily available to maintain the cognitive function of shift workers.
These ndings indicate that the subjects who ate a small meal felt less hungry for 5 h (i.e., until 09:00), and that those who did not eat at night perceived greater hunger toward morning. Although these ndings may predominantly re ect a longer inter-meal interval, hunger is known to display a circadian rhythm and be increased during the night. Restricted sleep also increases hunger and appetite, and results in a preference for a high-energy diet rich in carbohydrates [38]. Night work has been reported to cause mental stress [39], which can trigger fatigue and lead to decreased performance [40,41]. As short-term changes in food intake are known to affect various aspects of cognitive function, we believe that reducing the stress induced by hunger may help sustain performance. Compared with high-fat meals, those rich in carbohydrates reduce mental (in contrast to physical) performance and increase sleepiness [42]. Eating a heavy lunch is associated with signi cant increases in motor vehicle accidents and a tendency to experience greater subjective sleepiness [43]. More generally, objective signs of sleepiness typically peak about 3.5 h after eating [44]. In other words, in this respect, tasks that require sustained attention are the most sensitive, with larger meals producing more frequent lapses of attention [45]. However, in the present study, the consumption of two rice balls (containing approximately 352 kcal of primarily carbohydrates) did not appear to affect sleepiness and fatigue.
In the present study, the heart rate was signi cantly higher under the snack than under the Skipping condition from 04:00 to 06:00. When eating a meal, visceral circulating blood volume increases to support the process of digestion and absorption, the sympathetic nervous system supports it, and the heart rate increases [46]. The contribution of increased sympathetic nervous system activity postprandially to the thermic effect of food is not always evident, and it has been shown to depend on the size and composition of the meal, with the clearest effect seen with carbohydrates. The brain integrates signals related to food intake from various sites (e.g., gut, hepatoportal area, chemoreceptors), leading to increased peripheral sympathetic out ow [46]. Few studies have focused on the relationship between meals and autonomic nervous system activity. The results of the present study showed that a small meal increased the heart rate and decreased parasympathetic nervous system activity, which is related to rest and digestive activity. The two systems, the sympathetic nervous system and the parasympathetic nervous system, generally act in a complementary fashion, because the increase in one is usually associated with a decrease in the other. Many sympathetic nervous system functions are opposite to those of the parasympathetic nervous system. In the present results for the low-frequency power/high-frequency power ratio values between 02:00 and 09:00, the Skipping condition showed no change, but the Snack condition resulted in an immediate decrease after eating. Because the lowfrequency power/high-frequency power ratio has been shown to represent changes in sympathetic nervous activity in the autonomic nervous system as a stress index [47], stress levels may have been lower under the eating condition. Consuming a meal is reportedly effective for reducing stress [48], which is consistent with the results of the present study. Among night shift nurses who remain awake for a long period of time, sleepiness and fatigue increase, and stress levels also gradually rise with increased workloads around the end of their shifts [49]. If stressful situations are prolonged, night shift workers may develop chronic fatigue. The results of this study suggest that eating a small meal at 03:30 may be effective for reducing stress in the morning. In addition, shift workers often report that increased alertness is one of the main factors that in uences their decision to eat during a shift [10]. Dietary strategies and altered eating behaviors have been reported as strategies to maintain alertness during the night shift among a sample of nurses [50]. Furthermore, reduced meal sizes have been reported as a strategy used by nurses to minimize gastric symptoms during night shifts. Therefore, promoting the consumption of a small meal at 03:30 during break time may help night shift workers maintain their health and improve safety during 16-h night shifts, as well as prevent malpractice and chronic fatigue.
In the case of long night shifts, the time for taking of meals is restricted, and it is di cult to eat freely. We recommend a small snack to maintain performance and stop the increase in errors regardless of time.
Rice balls, which can be easily eaten even when busy, may be one of the options for meals during night shifts.

Limitations
This study has several limitations. First, it was conducted under laboratory conditions; therefore, the degree of change in performance and sleepiness could vary from that under actual working conditions, which greatly depend on differences in workload due to the nature of the work or the timing of busy work periods. To con rm effective measures for reducing sleepiness and fatigue tailored to the conditions of speci c professions and workplaces, intervention studies need to be conducted in actual workplaces. It is possible that the present results would be different if actual shift workers had been studied; therefore, caution is needed when interpreting the results. Second, the participants in this study were all young women in their 20s with no shift work experience, which may have affected the results. We would like to investigate this issue further in a future study.
A previous study reported that taking a nap during a night shift helped nurses recover from night shift fatigue [51]. Therefore, in the future, it will be necessary to take measures to reduce drowsiness and fatigue by combining meals and naps.

Conclusions
This study investigated the effects of eating a meal at night during night shifts on subjective sleepiness, vigilant attention, processing speed, hunger, and autonomic nervous system activity between 16:00 and 09:00. The results showed that consuming a small meal (352 kcal) at 03:30 during the night shift reduced hunger and helped sustain performance until morning. To the best of our knowledge, this is the rst study to explore the effects of consuming a small meal at night on 16-h performance in a controlled laboratory environment.  Outcomes regarding the participants' physiological parameters, cognitive performance, and autonomic nervous system activity from 16:00 to 09:00 Electrocardiogram data could not be obtained from one participant during the experiment