Participant recruitment and ethical approval
Participants in this cross-sectional study were recruited from the students and staff of Sunway University and Sunway College, Sunway City, Selangor, Malaysia, from June – December 2022 (without COVID-19 movement restrictions) by convenience sampling, through publicity materials around campus and word-of-mouth. Participants must meet the following inclusion criteria: 1. Malaysian, aged 18 - 50 years; 2. no current major medical condition (e.g., cancer, liver or kidney disease); 3. no history of or current endocrine pathology (Cushing syndrome, pseudohypoparathyroidism, etc.); 4. no history of neurological disorder or injury (e.g. stroke, or seizures; loss of consciousness > 10 minutes); 5. no history of or current serious psychological disorder (i.e., severe depression or anxiety, substance use disorder, psychoses, bipolar disorder); 6. not currently pregnant or breastfeeding; 7. no impaired sensory function (e.g., visually impaired); 8. no physical activity contraindication; 9. not taking any medication that impacts weight and appetite (e.g., mirtazapine, prednisone); 10. no history of syndromic obesity (Prader Willi, Alström, Laurence-Moon Biedle syndrome, etc.). Screening of the inclusion criteria was performed during the participant’s first visit and if eligible, participants were assigned a subject ID. Briefing on how to answer the online questionnaires was performed, clinical, and anthropometric measurements were taken, and activity wristbands (Xiaomi® Mi Smart Band 5®) were loaned out. Two weeks later, an exit interview was performed where participants returned the activity wristbands, and were given a reimbursement.
Using the Raosoft sample size online calculator (http://www.raosoft.com/samplesize.html), a minimum sample size of 195 is needed to achieve a 7% margin of error, 95% confidence level, Sunway University and Sunway College population size of 22,000, and a 50% response distribution.
Ethical approval was obtained from the Sunway University Research Ethics Committee (SUREC 2022/008), all participants signed informed consent forms, and the study was conducted in accordance with the Declaration of Helsinki.
Sociodemographic and lifestyle factors questionnaire
Sociodemographics, i.e. self-identified Malaysian ethnicity (Malay/Chinese/Indian), age, highest education level (primary/secondary/tertiary), marital status (single/married/divorced or widowed) and monthly household income (B40/M40/T20). According to the Department of Statistics Malaysia (2019), monthly household income is defined as total gross income before taxes, received by all members of a household [for students, unemployed or financially-dependent individuals: parents' household income; for employed and financially-independent individuals: the combined (own, spouse's, children's household income)][12]. The B40, M40 and T20 categories were ≤ MYR4,850, 4851-10,960, and ≥ 10,961 (approximately ≤USD1065, 1066 – 2,406, and ≥2407), respectively [12].
Chronotype and sleeping behaviors
Chronotype was assessed using the 5-item reduced Horne-Ostberg Morningness-Eveningness (rMEQ) questionnaire [13]. Total scores of the 5-item rMEQ range from 4 to 26, whereby a higher score indicates a morningness chronotype. The same cutoff scores for determining chronotype groups were used as in [13](evening: < 12; neither: 12–17; morning: > 17).
Sleep behavior data were tracked via the Zepp Life® app (iOS® and Android®) using the Xiaomi® Mi Smart Band 5 (SKU: BHR4215GL). The Xiaomi® Mi Smart Band 5 has a 3-axis accelerometer, a three-axis gyroscope, a heart rate sensor, and a photoplethysmography sensor to measure some biomedical parameters including sleep behaviors. This activity wristband was chosen because it has been validated in a clinical trial (ClinicalTrials.gov NCT04568408), and was found to have an overall 78% accuracy, 89% sensitivity, and 35% specificity compared to the polysomnography (PSG) gold standard [14]. Albeit, we are aware of the limitations of this activity wristband, in the sense that it is more accurate in detecting wake (48%) and light sleep (51%), rather than in identifying deep sleep (34%) and REM sleep (28%) [14]. It also tends to misidentify PSG sleep phases 40% to 70% of the time, misclassifying 46% of the wake and 65% of the REM sleep stages as light sleep [14].
Participants were instructed to wear the wristbands throughout the day and night, during sleep, in either wrist, at approximately a finger width away from their wrist bone and tightness that allows direct skin contact with the back. They were requested to download the Zepp Life® app, pair the band via Bluetooth, and turn on the “Automatic heart rate monitoring & sleep assistant” setting in the app to enable more precise sleep data monitoring. Participants were asked to report via screen capture of the Zepp Life® app on the following parameters: fall asleep time, wake up time, duration of light, deep, REM and time awake during sleep, for any two weekdays and one weekend within a week. Participants who failed to wear the wristband throughout the day and night, as detected by the invalid/blank or abnormally short data, were excluded from analysis. An average of the aforementioned durations was calculated between two weekdays (average weekday), and between two weekdays and one weekend (overall average). Total Sleep Period is the summation of light, deep and REM durations.
Dietary Records and Chrononutrition Behaviors
Participants were instructed to record all foods and beverages consumed for three 24-hour periods, each day starting at 12:00 am and ending at 11:59 pm, for any two weekdays and one weekend within a 7-day week cycle. Specific details that need to be recorded included: time of meal consumed, place consumed (home, campus, name of restaurant, etc.), and the type of eating occasion or meal (breakfast, lunch, dinner, snack, or other), list each food/beverage item consumed, including foods eaten between meals and all drinks, even if it is a non-caloric item like water, coffee, tea, or sugar free gum, specific details, ingredients, preparation, brand name of each food or beverage consumed, and portion sizes of each food or beverage consumed, using the “Food Amounts Booklet” [15]. The amount of calories consumed for breakfast, lunch, and dinner occasions were estimated based on the Malaysian Food Composition Database (https://myfcd.moh.gov.my/myfcdcurrent/) and Singapore Energy and Nutrition Composition of Food Database (https://focos.hpb.gov.sg/eservices/ENCF/).
“Breakfast” was defined as recording their first meal before 1200, “lunch” as recording the second meal between 1201 to 1700, and “dinner” as recording the third meal between 1701 to 2359 within a 24-hour day. Meal skipping was defined as non-record of the meals taken at the above times.
Based on the dietary and sleep records, the following (average) weekday or weekend chrononutrition behaviors were calculated [16–18]: Breakfast/lunch/dinner jetlag = Breakfast/Lunch/Dinner time on weekends - Breakfast/Lunch/Dinner time on weekdays; Eating midpoint = ([Timing of the last meal - Timing of the first meal]/2) + Timing of the first meal; Eating jetlag = Eating midpoint on weekends - Eating midpoint on weekdays; Eating window = Last eating event before bedtime – First eating event time; Weekly average eating window (weighted mean) = [(2 × eating window on weekdays) + (1 eating window on weekends)]/3; Sleep duration = Wake time – Fall asleep time; Morning latency = First eating event time – Wake time; Lunch latency = Lunch time – First eating event time; Afternoon latency = Last eating event before bedtime – Lunchtime; Evening latency = Fall asleep time - Last eating event before bedtime; Sleep midpoint = Fall asleep time + (Sleep duration/2); Social jetlag = sleep midpoint weekdays – sleep midpoint weekend.
Since the NutriNet-Santé study showed that having a later first meal (later than 0900 compared to earlier than 0800) and last meal of the day (later than 2100 compared to earlier than 2000) were associated with a higher risk of cardiovascular outcomes [19], we also determined the frequencies of the timings of first meal - before 0800, 0800-0900, and after 0900; the timings of last meal - before 2000, 2000-2100, and after 2100; and the durations of nighttime fasting (24 h minus the time elapsed between the first and the last meal of the day) – < 12h or less, 12-13 h, > 13 h.
Integer values of social, eating, breakfast, lunch, or dinner jetlag were used to evaluate the frequency of the delay or advance of each sleep or meal timing on weekends. Thereby, “advance” in the timing of a sleep/meal was considered if values were lower than -1, “delay” in the timing of a sleep/meal was considered if values higher than +1, and the “maintenance” in the timing of the sleep/meal was considered if values ranged from -1 to +1 [18]. For example, advance in sleep/meal time would be considered as sleeping/eating two hours earlier on weekends while delayed sleep/meal time indicated eating two hours later on weekends. Maintenance meant sleeping/eating at the same time on weekdays and weekends.
The day(s) of breakfast skipping was/were extrapolated by multiplying the total number of two weekdays and one weekend where breakfast time(s) was/were not recorded by 2.33. The largest meal is defined as the meal (breakfast, lunch or dinner) in which largest amount of calories are consumed.
Measurement of appetite sensations
The visual analogue scale (VAS), 100mm in length with words anchored at each end, expressing the most positive and the most negative rating, were used to assess hunger, satiety, fullness, prospective food consumption, desire to eat something fatty, salty, sweet or savory [20]. Participants were instructed to write down the approximate time the meal was consumed, and to rate for at least 3 meals consumed for three 24-hour time periods, each day starting at 12:00 am and ending at 11:59 pm, for any two weekdays and one weekend within a 7-day week cycle.
Clinical, anthropometric and body composition measurements
Clinical measurements indicative of vascular health namely systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse rate were taken using an automated blood pressure monitor (HEM-7121, Omron, Japan) after the subjects had rested for 5 min. Height was measured using a wall-mounted stadiometer. Waist and hip circumferences were measured using a stretch-resistant tape that provided a constant 100 g tension, at the midpoint between the lower margin of the least palpable rib and the top of the iliac crest and around the widest portion of the buttocks, respectively [21]. The waist-hip ratio (WHR) and waist-to-height ratio (WHtR) were calculated by dividing waist circumference by hip circumference and height, respectively. A bioimpedance body composition scale (Omron HBF-375) was used to determine weight, body mass index (BMI; kg/m2), total body fat (TBF; %), visceral fat level (VFL; %), subcutaneous fat (SF; %), skeletal muscle percentage (SM; %) and resting metabolism rate (RM; kcal). The cutoff points for overweight, obesity, high TBF, high VFL, high SM, high WC, high WHR and high WHtR are ≥23 kg/m2 [22]; ≥27.5 kg/m2 [22]; 20% (men) or 30% (women) [23]; 10% [23]; 35.8% (men) or 28% (women) [23]; 90 cm (men) or 80 cm (women) [22]; 0.90 (men) or 0.85 (women) [21]; and 0.50 [24], respectively.
Statistical analysis
Statistical analysis of the data was performed using IBM SPSS Statistics for Windows 26.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics for the categorical variables (demographic characteristics) were presented in terms of frequency and percentage. The conformity of the numerical variables to normal distribution was determined by the Kolmogorov-Smirnov test, where p > 0.05 indicates normally-distributed data. Pearson chi-square test was used to test the differences in categorical variables of demographic, adiposity status, chronotype, social jetlag, and chrononutrition behavior classes between genders. Mann–Whitney U test (U) was used in the comparison of two independent groups that did not have a normal distribution, while the Kruskal–Wallis test was used in the comparison of more than two groups. Multiple linear regression was conducted to identify the anthropometric and body composition measurements associated with chrononutrition behaviors. All assumptions for multiple linear regression were fulfilled and the models were controlled for sociodemographic factors: gender, ethnicity, age, marital status, highest education level and monthly household income. Examination of the relationships between the scales was determined by the Spearman rank differences’ correlation coefficient. In the interpretation of the correlation coefficient, it was determined as a “very weak correlation, if <0.2”, a “weak correlation between 0.2 and 0.4”, a “moderate correlation between 0.4 and 0.6”, a “high correlation between 0.6 and 0.8”, and “0.8 > very high correlation”. The p-value of < 0.05 was considered statistically significant.