The impact of Ramadan fasting on glucose variability in type 2 diabetes mellitus patients on oral anti diabetic agents.

Ramadan fasting is associated with changes in eating, physical activity, sleeping patterns, and medication. Unfortunately, only limited studies examine glucose variability in subjects with type 2 diabetes who fast in Ramadan. Our study aims to evaluate glucose variability in subjects with type 2 diabetes on oral antidiabetic agents using continuous glucose monitoring system (CGMS) during and after Ramadan fasting. This observational study was done in The Indonesian Medical Education and Research Institute, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia, which recruited 10 subjects with type 2 diabetes who underwent Ramadan fasting in 2019. These subjects were free from cardiovascular disease, kidney disease, severe liver disease, chronic gastrointestinal disease and autoimmune disease. Insertion of CGMS for measuring interstitial glucose was performed after at least 2 weeks of Ramadan fasting and 4 weeks after the end of the Ramadan fasting, with a minimum of 3 days observation. The mean amplitude of glycemic excursion (MAGE) during and after Ramadan were similar (p = 0.94). In line with this, the average interstitial glucose (p = 0.48), the maximum interstitial glucose (p = 0.35), the minimum interstitial glucose (p = 0.24), and the duration of hypoglycemia (p = 0.25) were also similar in both periods. Overall, nutritional intake and energy expenditure during both periods were comparable. Ramadan fasting is not associated with increased glucose variability in subjects with type 2 diabetes. Thus, Ramadan fasting is safe in subjects with type 2 diabetes with no complications.

Introduction

Fasting in Ramadan is one of the five pillars of Islam. [1,2] Muslims are not allowed to eat, drink, smoke, and even take their medication from predawn (suhur) to sunset (iftar) every day during Ramadan month. [3] Fasting during Ramadan is not obliged for Muslims with serious illness, including for certain people with diabetes. [4,5] Unfortunately, some people with diabetes who are advised not to fast in Ramadan persist in fasting during Ramadan. [6-10]

Ramadan fasting is associated with changes in eating, physical activity, sleeping patterns, and medication which potentially lead to an increasing rate of hypoglycemia and hyperglycemia. [11-13] Glucose variability is considered as a major determinant of hypoglycemia or hyperglycemia risk, which is coherently linked to extreme glucose fluctuations. [14] Higher glucose variability is also linked to the development of microvascular and macrovascular complications. [15-18] Thus, glucose variability, aside from HbA1c, should be considered as one of the important targets in type 2 diabetes management, especialy during the Ramadan month. [3,15,19]

In order to acquire the data about glucose variability during Ramadan, more frequent self-monitoring of blood glucose levels (up to 7 times) is recommended, especially in the subjects with history of symptomatic hypoglycemia or hyperglycemia. [5] However, this practice is related to inconvenience, related to multiple and painful finger pricks. [20] Continuous Glucose Monitoring System (CGMS) is a minimally invasive blood glucose examination that produces the mean amplitude of glycemic excursion (MAGE) index, which is considered as the gold standard for glucose variability. [3,16,21] CGMS uses a sensor that is subcutaneously inserted to obtain a continuous record of interstitial glucose levels. [22-24] CGMS has been widely used in clinical and research settings to assess glucose variability in subjects with diabetes. For this reason, CGMS is considered useful for evaluating glucose variability during Ramadan fasting. [3] However, only limited studies had investigated the glucose variability in type 2 diabetes during Ramadan. [1-3,9,25,26]

Our study aims to evaluate the glucose variability of subjects with type 2 diabetes during and after Ramadan fasting using CGMS. We will also assess whether changes in diet and physical activity contributed to the changes in glycemic variability. Furthermore, the rate of hypoglycemia and hyperglycemia will also be assessed.

Materials and methods

Design and ethics

This prospective longitudinal cohort study was conducted at the Metabolic Disorder, Cardiovascular and Aging Cluster of The Indonesian Medical and Education Research Institute (IMERI), Faculty of Medicine Universitas Indonesia (FKUI), in May–July 2019. Ethical approval was obtained from the FKUI (Protocol Number: 18-04-0523). The CGMS procedure and study protocol were explained to the subjects and written informed consent was attained.

Subjects selection

Subjects with type 2 diabetes who participated in this study were recruited from various health facility centers around Jakarta using a consecutive sampling method. Subjects with previous documentation of cardiovascular disease, kidney disease, severe liver disease, chronic gastrointestinal disease, and autoimmune disease were excluded from this study. This study only included subjects who had completed Ramadan fasting for a minimum of 14 days and had consented to come back one month after Ramadan ends for another CGMS examination. The subjects who failed to complete the second visit will be excluded from analysis.

Continuous glucose monitoring system

The Medtronic iPro®2 CGMS was used to perform continuous glucose monitoring, with a minimum observation period of 3 days. The CGMS sensor collects the interstitial glucose (interstitial glucose) records at 5 minutes intervals for a total of 288 readings every 24 hours. In addition to CGMS, the subjects were also instructed to simultaneously perform self-monitored blood glucose (SMBG) using Roche Accucheck Performa Glucometer® for calibration of interstitial glucose measurement obtained from CGMS. This SMBG was performed every day during the CGMS monitoring, twice per day, before suhur (on fasting day) or fasting blood glucose (on the period after Ramadan) as well as at any time before bed during both periods. They were also instructed to do SMBG if they experience hypoglycemia or hyperglycemia symptoms. These symptoms were explained before all subjects had the CGMS inserted. Following insertion day, all subjects were instructed to come back after 7 days for sensor removal. The data were downloaded using Medtronic Minimed Software from the sensor with a measuring range between 40 to 400 mg/dL. [27]

MAGE was set as the primary outcome of this study, which was calculated using the formula by Kovatchev et al. [14] We also calculated average, minimum, and maximum interstitial glucose. The average interstitial glucose readings of all subjects at the same time points during the examination were extracted and described in the curve with a 3-hours interval, representing fasting and non-fasting curve. Besides MAGE and other CGMS parameters, we also compared the incidence rate of symptomatic hyperglycemia and hypoglycemia. We set the target of blood glucose within 70–150 mg/dL. Symptomatic hyperglycemia was defined as the measurement of blood glucose ≥200 mg/dL with hyperglycemic symptoms, while symptomatic hypoglycemia was defined when the occurrence of hypoglycemic symptoms with blood glucose ≤70 mg/dL.

Anthropometry measurement

The body weight and body composition measurement were conducted using Tanita MC780MA bioimpedance analyzer (BIA), while a portable stadiometer (GEA Medical, SH-2A High Meter 2 M) was used to measure height. Waist circumference was measured using an ergonomic circumference measuring tape based on WHO standard protocol, as the middle point between the last palpable costae and the top of illiac crest. [28] The blood pressure measurement was done in sitting position after resting for 10–15 minutes using GEA Medical® type SH-2A High Meter 2 M.

Nutritional intake measurement

The nutritional intake data were attained using a 3-day non-consecutive food record, of which all subjects were asked to write their food and drink consumption for two days during the weekday and one day during the weekend. The food record data were then verified by a certified nutritionist at the time when the CGMS sensor was disconnected. Nutritional analysis was then performed using Nutrisurvey® program. The final nutritional data was obtained after calculating each average parameter value and these data were then displayed in the table.

Physical activity measurement

The physical activity data were assessed using Bouchard questionnaire [29] and performed by the subjects at home. This questionnaire captured the activity of each subject every 15 minutes for 24 hours, resulting in 96 periods, and was also performed for two days during the weekday and one day during the weekend for each visit. For each 15 minute periods, the subjects were instructed to fill it with a number, ranging from 1–9, according to the intensity of the predominant activity during that period.

The results of the questionnaire will be quantified to yield energy expenditure by which will be depicted by Metabolic Equivalent (METs) in kcal/kg. The final energy expenditure data were obtained by counting the average of METs every 3 hours during 3-day courses. The data were further transferred into a graph representing 24-hour METs during and after Ramadan fasting.

Laboratory measurement

Whole blood samples were collected using EDTA vacutainer (GP Vacuum Tube) after 10–12 hours of fasting and were used for HbA1c measurement using standardized High-Performance Liquid Chromatography (HPLC) method (Bio-Rad D-10 HbA1c Autoanalyzer). [30] The fasting blood glucose was measured using capillary blood using Roche Accucheck Performa Glucometer®.

Data analysis

The comparison analysis of the primary and secondary outcomes during and after Ramadan was conducted using paired T-test for normally distributed data and Wilcoxon test for non-normally distributed data. All of the normally distributed data were displayed in the mean with standard deviation, whereas the non-normally distributed data were displayed in the median with interquartile range. All analyses were performed using SPSS program version 20 (IBM Statistics).

Results

Baseline characteristics

Ten subjects with type 2 diabetes were recruited in this study. Four weeks after Ramadan fasting, two subjects were discontinued from the study due to poor compliance. The flowchart of study timeline is shown in S1 Fig.

The baseline characteristics of the 10 subjects are summarized in Table 1. The mean value of HbA1c was 8,8 (2,8)% or 73 mmol/mol. All subjects consumed metformin in which four subjects were given metformin only, while the other six were in combination with other oral anti-diabetic drugs (OADs). Five subjects were given metformin and sulphonylurea (SU) combination while the other one was given metformin, SU, and α-glucosidase inhibitor. During Ramadan fasting, there was no change in the total dose of OADs, only the timing of OADs administration was modified. Subjects who were given metformin consumed 500 mg at suhur time and 1000 mg at iftar time. Meanwhile, subjects who were given SU as combination consumed only at iftar time.

Table 1

Baseline characteristics.

Parameter	Subjects (n = 8)
Age (year, mean, SD)	52,5 (6,3)
Male (n,%)	4 (50)
Duration of Diabetes (year, mean, SD)	3,9 (4,3)
Family History of Diabetes (n,%)	6 (75)
Hypertension, (n,%)	4 (50)
Dyslipidemia, (n,%)	2 (25)
Systolic Blood Pressure (mmHg, median, IQR)	120,0 (102,5–130,0)
Diastolic Blood Pressure (mmHg, median, IQR)	80,0 (77,5–82,5)
Waist Circumference (cm, mean, SD)	90,0 (11,5)
AST (u/l)	24 (10–57)
ALT (u/l)	17.88 (7–45)
Fasting Blood Glucose (mg/dL, mean, SD)	151,0 (63,7)
HbA1c (mmol/mol, mean, SD)	73 (7)
Weight (kg, mean, SD)	64,5 (13,5)
Body Mass Index (kg/m2, mean, SD)	25,9 (6,1)
Medication
1 OAD (n%)	4 (50)
2 OADs (n%)	3 (37,5)
≥ 3 OADs (n%)	1 (12,5)

OAD: oral anti diabetic drug

Glucose variability data

As the primary outcome of this study, MAGE values in the fasting and non-fasting period were similar (p = 0.94) (Table 2). In line with this, the average interstitial glucose (p = 0.48), the maximum interstitial glucose (p = 0.35), the minimum interstitial glucose (p = 0.24), and the rate of hypoglycemia (p = 0.25) were also similar in both periods (Table 2). Interestingly, the percentage of interstitial glucose within target appeared higher during Ramadan compared to after Ramadan period, but it did not reach statistical significance (37,6% v.s 26,9%, p = 0.27). Additionally, the percentage of interstitial glucose values above the target was relatively high, especially in the non-fasting period (72,4% v.s 60,4%, p = 0.26). There was no incidence of symptomatic hyperglycemia or hypoglycemia during this study.

Table 2

CGMS results.

CGMS Parameter (subject n = 8)	Ramadan	After Ramadan	p value
Total reading (times)	14 413	14 003	0.89
Mean IG (mg/dL, mean, SD)	191 (30)	203 (16)	0.48
SD IG (mg/dL, mean, SD)	36 (7)	46 (8)	0.11
Maximum / Highest IG (mg/dL, mean, SD)	321 (28)	339 (19)	0.35
Minimum / Lowest IG (mg/dL, mean, SD)	80 (21)	89 (15)	0.24
IG above target (%, mean, SD)	60.4 (30.9)	72.4 (25.9)	0.26
IG within target (%, mean, SD)	37.6 (29.6)	26.9 (26.2)	0.27
IG below target (%, median, IQR)	1.0 (0–4.5)	0 (0–1.5)	0.25
MAGE (mg/dL)	6.75	6.58	0.94

IG: interstitial glucose; SD: standard deviation; MAGE: mean amplitude of glycemic excursion, p value < 0.05 is considered statistically significant

Despite there was no difference in the MAGE value, we observed a notable interstitial glucose fluctuation during Ramadan fasting compared to the non-fasting period (Fig 1A). The peak of interstitial glucose during Ramadan fasting was observed at 6 AM, while after Ramadan, the peak was found at 12 AM. The interstitial glucose reached the lowest point at 6 PM during Ramadan (just before iftar), while during the non-fasting period, the lowest point was at 6 AM (Fig 1A). Moreover, when we compared pairs of different timepoint, there were significantly difference at 3 AM (p = 0,012), 6 AM (p = 0.027), and 6 PM (p = 0.012). (S3 Table) When we grouped the subject into 2 groups; group 1 as subjects who only took metformin and group 2 as subjects who had combination of metformin and other OADs (SU or acarbose) in their regimens, the pattern of interstitial glucose during fasting and non-fasting period were similar in both groups (S2 and S3 Figs). Furthermore, when we compared the pattern of interstitial glucose during fasting and non-fasting between male and female, there were no significantly differences.

Fig 1

Comparison of 24-hour interstitial glucose levels and energy expenditure in METs during and after Ramadan.

The levels of interstitial glucose (A) and METs (B) are presented as mean (SD) of all interstitial glucose readings and energy expenditure in every 3 hours period. The fasting period is depicted as the black dot, while the non-fasting period is depicted as the white square.

Nutrition intake and physical activity profile

The pattern of diet in our subjects during the fasting and non-fasting period was similar (S1 Table). If anything, we observed a slight higher total energy intake during the Ramadan fasting period, which might be contributed by the higher carbohydrate intake (S1 Table). When comparing the nutrition intake during suhur and iftar, there was a trend for a higher total energy intake and carbohydrate intake during iftar. (Fig 2, S2 Table). The physical activity profiles during the fasting and non-fasting period were comparable (Fig 1B).

Fig 2

Total energy intake during suhur and iftar in Ramadan.

The levels of total energy intake are presented as mean (SD) during suhur and iftar. The suhur period is depicted as the black dot, while the iftar period is depicted as the black square.

Discussion

Our study has demonstrated that glucose variability in type 2 diabetes did not differ significantly between during the fasting Ramadan period and non-fasting period after Ramadan. Other parameters such as average, highest, and lowest interstitial glucose were comparable. There was no difference in hypoglycemia rate between two periods. In addition, there was no incidence of symptomatic hypoglycemia and hyperglycemia. This implies that fasting in Ramadan does not increase the likelihood of glucose variability in subjects with type 2 diabetes. However, interstitial glucose fluctuation was observed, but it did not reach statistical significance.

The main finding of this study confirmed that glucose variability, as assessed using MAGE, during the fasting Ramadan was not significantly different compared to non-fasting period. Our findings added important data to the current limited studies assessing MAGE during Ramadan fasting in type 2 diabetes using CGMS devices which reported conflicting results. [3,25] Our study also confirmed previous studies that reported overall CGMS parameters such as average, maximum, and minimum interstitial glucose were not significantly different between the two periods. Many factors have been linked to the variation of glucose variability in subjects with type 2 diabetes which involve the alteration of diet pattern, physical activity and medication timing. [3]

It is important to note that previous studies did not assess nutritional intake and physical activity profile related to changes in CGMS parameters. [1,3,26] While both previous studies by Lessan et al. and Aldawi et al. lacked nutritional and physical activity data, our study managed to fill the gap by presenting a complete profile of both domains. We observed that total energy intake did not differ between both periods, as well as other dietary parameters. Furthermore, the physical activity profile, represented by METs, was also comparable. These overall similar profiles in term of diet and physical activity might contribute to the fact that we observed no changes in glucose variability in the fasting period. The changes in anti-diabetic agents consumption during Ramadan fasting might also influence the glucose variability in this period. [3] The medication timing is usually taken in a higher dose during iftar, whether taken before or after breaking the fast. [5] Sulphonylurea (SU) is also associated with a higher incidence of hypoglycemia during Ramadan fasting. [31] However, our study showed that the changes of medication timing and type of medication did not seem to affect overall glycemic variability during the fasting Ramadan.

While Ramadan fasting was not associated with changes in overal glycemic variability, it was associated with a marked glucose surge after iftar. The glucose surge after iftar is mainly caused by the common practice of eating food with high glucose content such as dates. [3,5] Different culture will certainly affect the pattern of food that are consumed during iftar, but the common ground is the consumption of dates to break the fast. [32-34] Following the food that is eaten to break the fast, people will eat more food either before the practice of tarawih (night prayer conducted only in Ramadan) or afterward that will contribute to the surge of blood glucose level. [35,36] Our study indeed showed that carbohydrate consumption was higher during iftar compared to suhur.

Other important findings in this study are the information about the timing of the lowest interstitial glucose occurence. Despite reaching the nadir at the end of the fasting period, just before iftar, our study showed a very low percentage of asymptomatic hypoglycemia event which was comparable to the the non-fasting period. Our finding was not in line with studies by Bonakdaran et al. [1] and Alawadi et al. [26] which highlighted an increased event of hypogylcemia during Ramadan. The contrasting result might be explained by the fact that our study subjects have a higher baseline HbA1c and the exclusion of subjects with kidney disease, in contrast to the previous studies.

Despite being the first study in type 2 diabetes that presents the CGMS parameters along with nutritional intake and physical activity profiles during Ramadan fasting and afterward, our study has several limitiations. The number of subject in our study was relatively small and only recruited low or moderate risk subjects. Besides, we used 3 non-consecutive days for food consumption and activity record instead of 7 days record.

In summary, despite the post-iftar surge of glucose level after previously reaching the nadir just before the iftar, the overall glucose variability in subjects with type 2 diabetes who undergo Ramadan fasting does not differ during and after Ramadan, thus, confirming the safety of Ramadan fasting for subjects with type 2 diabetes. It is also important to note that the pre-iftar period is a period with the highest risk of hypoglycemia, thus it is advisable for subjects with type 2 diabetes to perform a more frequent blood glucose checking during that period. Furthermore, with a notable surge of glucose after iftar, adjustment in term of dietary intake and medication dosage should be recommended.

Flow chart of study timeline.

(TIFF)

Click here for additional data file.

Comparison of interstitial glucose levels during Ramadan based on medication group.

(TIF)

Click here for additional data file.

Comparison of interstitial glucose levels after Ramadan based on medication group.

(TIF)

Click here for additional data file.

Comparison of dietary compositions during Ramadan and after Ramadan.

(DOCX)

Click here for additional data file.

Comparison of dietary compositions during suhur and iftar.

(DOCX)

Click here for additional data file.

Comparison of interstitial glucose during Ramadan and after Ramadan.

(DOCX)

Click here for additional data file.

31 Mar 2020

PONE-D-20-05586

The impact of ramadan fasting on glucose variability in type 2 diabetes mellitus patients

PLOS ONE

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Reviewer #1: Ramadan is the 9th month of the Islamic lunar calendar, during which Muslims abstain from food intake between sunrise and sunset. Major changes to dietary patterns occur during Ramadan as meals are taken before dawn and immediately after sunset. In the present trial Harbuwono et al. compare glucose variability in patients with type 2 diabetes using CGMS during and after Ramadan fasting.

Thank you for the opportunity to review this interesting work. Following are some notes:

Please use 'subjects with type 2 diabetes' instead of 'type 2 diabetes subjects' throughout the manuscript.

L74: please add citations

L78: typing error – also

Design and ethics

Please use prospective longitudinal cohort study fort he study design. Comparative generally describes comparison of groups, which is not correct in this trial.

Did the participants give written informed consent before they were included in the trial?

Was the study apporved by the University or by a ethics committee oft he University? Please clarify and add the ethics number.

Was the trial registered in a registry for clinical trials (eg clinicaltrials.gov)? Please add information.

Subjects selection

Please describe exclusion criteria (kidney disease, severe liver disease) in more detail, based on lab measurements (ASAT, ALAT , eGFR,..).

Were patients with insulin or GLP-1 therapy also excluded?

Are there any limitation regarding HbA1c? Please clarify.

Anthropometry measurement

Please add information about blood pressure measurement.

Results

L163ff: Only eight subjects were included in the final analysis – please change also the baseline characteristics (table 1) to these eight subjects.

Table 1

Please add HbA1c in mmol/mol

Glucose variability data

L177: the rate of hypoglycemia (p=0,25) were similiar in both groups – where are the numbers of hypoglemic events? Please add these values (in table 2)

L181 vs. Table 2: 74,2% IG above target in the text versus 72,4% in table 2, please clarify.

Table 2: please add number of subjects included in this analysis.

Please use mean IG instead of average IG

L189: ..the peak was found at 3PM, please check.

L191: please update the analysis for 8 patients.

L195: Is this the figure legend to figure 1?

Figure 1A:

Were there some differences statistically significant at diverse timepoints?

Figure 2:

Please change axis title to „energy intake“

Figure S1:

Baseline characteristics can only be assessed once in a trial, please remove at T2 (4 weeks after Ramadan).

Maybe it would be more correct to illustrate 4 time points in this figure, since the CGMS disconnection are again 2 independent time points.

Anthropometric data and laboratory data were also collected at timepoint 2, please include them in the results section.

Reviewer #2: Dante Saksono Harbuwono et al. present a paper on the impact of fasting during and after Ramadan in type 2 diabetes mellitus patients. The paper is well written and the data has been thoroughly analysed. The observational study used a continuous glucose monitoring system (CGMS) to assess glucose variability during and after Ramadan but the sample size is small with only 10 people.

There are some major issues to adress:

1. It should be clarified in the title as well as the abstract that the focus of this paper is on patients with type 2 diabetes on OADs not patients with GLP-1 receptor agonists or insulin.

2. Why were only 3 non-consecutive days of food consumption and activity recorded?

3. How would the authors explain the significantly higher intake of fibre at iftar compared to suhur?

4. The patients chosen for this study are low to moderate risk and had a relatively high HbA1c level. They spent the majority of the time during CGMS above the interstitial target glucose. The safety of Ramadan fasting measured by glucose variability was shown for this specific group. Studies to confirm the safety of Ramadan fasting for type 2 diabetes subjects with a lower HbA1c or more time in the interstitial target glucose range are still necessary.

5. The small sample size is making it difficult to categorize subgroups but differences due to gender would be interesting.

As well as some minor issues:

1. It should be specified whether the informed consent was attained in written or in verbal form.

2. In the introduction line 66 CGMS is described as a minimally invasive blood glucose examination, whereas in the following sentence it is clearly stated it measures interstitial glucose levels which correlate with blood glucose levels. Also "interstitial glucose" is unnecessarily repeated in brackets.

3. Information on the time when the patients took the medication (morning and/or evening) and daily dosage should be added.

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Reviewer #2: No

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10 May 2020

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #2: Partly

Responses:

Reviewer 1 & 2: Thank you for the reviewer’s feedback.

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Reviewer #1: Yes

Reviewer #2: Yes

Responses:

Reviewer 1 & 2: Thank you for the review.

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer 1: Thank you for the review.

Reviewer 2: Thank you for the review.

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Reviewer #2: Yes

Responses:

Reviewer 1: Thank you for the review. We have corrected the typographical errors.

Reviewer 2: Thank you for the review.

Reviewer #1: Ramadan is the 9th month of the Islamic lunar calendar, during which Muslims abstain from food intake between sunrise and sunset. Major changes to dietary patterns occur during Ramadan as meals are taken before dawn and immediately after sunset. In the present trial Harbuwono et al. compare glucose variability in patients with type 2 diabetes using CGMS during and after Ramadan fasting.

Thank you for the opportunity to review this interesting work. Following are some notes:

Responses: We would like to thank the reviewer for careful and thorough reading of this manuscript and for his/her helpful comments.

Please use 'subjects with type 2 diabetes' instead of 'type 2 diabetes subjects' throughout the manuscript.

Responses: Thank you for the review. We have revised this section.

Unfortunately, only limited studies examine glucose variability in subjects with type 2 diabetes who fast in Ramadan. Our study aims to evaluate glucose variability in subjects with type 2 diabetes using continuous glucose monitoring system (CGMS) during and after Ramadan fasting.

This observational study was done in The Indonesian Medical Education and Research Institute, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia, which recruited 10 subjects with type 2 diabetes who underwent Ramadan fasting in 2019.

Ramadan fasting is not associated with increased glucose variability in subjects with type 2 diabetes.

Our study aims to evaluate the glucose variability of subjects with type 2 diabetes during and after Ramadan fasting using CGMS.

Subjects with type 2 diabetes who participated in this study were recruited from various health facility centers around Jakarta using a consecutive sampling method.

This implies that fasting in Ramadan does not increase the likelihood of glucose variability in subjects with type 2 diabetes.

Many factors have been linked to the variation of glucose variability in subjects with type 2 diabetes which involve the alteration of diet pattern, physical activity and medication timing.

In summary, despite the post-iftar surge of glucose level after previously reaching the nadir just before the iftar, the overall glucose variability in subjects with type 2 diabetes who undergo Ramadan fasting does not differ during and after Ramadan, thus, confirming the safety of Ramadan fasting for subjects with type 2 diabetes. It is also important to note that the pre-iftar period is a period with the highest risk of hypoglycemia, thus it is advisable for subjects with type 2 diabetes to perform a more frequent blood glucose checking during that period

L74: please add citations

Responses: Thank you for the review. We have revised this section. (page 4 Line 77-78)

However, only limited studies had investigated the glucose variability in type 2 diabetes during Ramadan.[1-3,9,25,26]

L78: typing error – also

Responses: Thank you for the review. We have revised this section. (page 4 Line 82)

Furthermore, the rate of hypoglycemia and hyperglycemia will also be assessed.

Please use prospective longitudinal cohort study for the study design. Comparative generally describes comparison of groups, which is not correct in this trial.

Responses: Thank you for the review. We have revised this section. (page 4 Line 86)

This prospective longitudinal cohort study was conducted at the Metabolic Disorder, Cardiovascular and Aging Cluster of The Indonesian Medical and Education Research Institute (IMERI), Faculty of Medicine Universitas Indonesia (FKUI), in May – July 2019.

Did the participants give written informed consent before they were included in the trial?

Responses: Thank you for the review. Yes, the subject gave written informed consent. We have revised to state that in the articles. (page 4 Line 90)

The CGMS procedure and study protocol were explained to the subjects and written informed consent was attained.

Was the study approved by the University or by an ethics committee of the University? Please clarify and add the ethics number.

Responses: We appreciate the reviewer’s feedback, but we already stated that The study was approved by the ethics committee of the university and the ethics number were inserted in the page 4 Line 89.

Ethical approval was obtained from the FKUI (Protocol Number: 18-04-0523).

Was the trial registered in a registry for clinical trials (eg clinicaltrials.gov)? Please add information.

Responses: Thank you for the review. No, we did not register this study for clinical trials.

Please describe exclusion criteria (kidney disease, severe liver disease) in more detail, based on lab measurements (ASAT, ALAT , eGFR,..).

Responses: Thank you for the review. Subjects with previous documentation of cardiovascular disease, kidney disease, severe liver disease, chronic gastrointestinal disease, and autoimmune disease were excluded from this study. The history of those diseases was obtained from careful history taking as well as the documentation of the several laboratory examinations. Subjects with an increased level of AST and ALT were considered to suffer liver disease and subjects with eGFR <90 were considered to suffer kidney disease. The history of chronic gastrointestinal disease and autoimmune disease were obtained only from history taking.

Were patients with insulin or GLP-1 therapy also excluded?

Responses: Thank you for the review. No, if patients with insulin or GLP-1 therapy meet the inclusion criteria, they will be included to the study. But, unfortunately, no patients with injectable therapy was included in this study.

Are there any limitation regarding HbA1c? Please clarify.

Responses: Thank you for the review. There is no limitation in HbA1c examination. HbA1c should be recorded every 3 months, but in this study we recorded after 1 months as representation of glucose level of the subjects.

Please add information about blood pressure measurement.

Responses: Thank you for the review. The blood pressure measurement was done in sitting position after resting for 10-15 minutes. We have revised to add this information in the article (page 6 Line 132).

The blood pressure measurement was done in sitting position after resting for 10-15 minutes using GEA Medical® type SH-2A High Meter 2 M.

L163ff: Only eight subjects were included in the final analysis – please change also the baseline characteristics (table 1) to these eight subjects.

Responses: Thank you for the review. We have revised the baseline characteristics (table 1) in the article.

Please add HbA1c in mmol/mol

Responses: Thank you for the review. We have now used the OAD acronym as oral antidiabetic drug throughout the manuscript. We also have changed HbA1c units from % to mmol/mol.

All subjects consumed metformin in which four subjects were given metformin only, while the other six were in combination with other oral anti-diabetic drugs (OADs).

L177: the rate of hypoglycemia (p=0,25) were similiar in both groups – where are the numbers of hypoglemic events? Please add these values (in table 2)

Responses: Thank you for the review. We have addressed this in table 2 as IG below target. In this study there was no symptomatic hypoglycemic event. But, according to IG parameter, there were some subjects whose IG below target.

Table 2: please add number of subjects included in this analysis.

Please use mean IG instead of average IG

Responses: Thank you for the review. We have revised this section.

Mean IG (mg/dL, mean, SD)

L189: ..the peak was found at 3PM, please check.

Responses: Thank you for the review. After we checked the data, the peak was found at 12 AM after Ramadan We have revised this section in the article. (page 10 line 279)

L191: please update the analysis for 8 patients

Responses: Thank you for the review. We have updated the analysis of 8 subjects in the article.

L195: Is this the figure legend to figure 1?

Responses: Thank you for the review. Yes, this is the figure legend to Figure 1A dan 1B.

Were there some differences statistically significant at diverse timepoints?

Responses: Thank you for the review. We have revised to add this information in the article. (page 10 line 281-283)

Moreover, when we compared pairs of different timepoint, there were significantly difference at 3 AM (p = 0,012), 6 AM (p = 0.027), and 6 PM (p = 0.012).

Please change axis title to „energy intake“

Responses: Thank you for the review. We have revised this section.

Baseline characteristics can only be assessed once in a trial, please remove at T2 (4 weeks after Ramadan).

Maybe it would be more correct to illustrate 4 time points in this figure, since the CGMS disconnection are again 2 independent time points.

Anthropometric data and laboratory data were also collected at timepoint 2, please include them in the results section.

Responses: Thank you for the review. We have revised out timepoint diagram.

Reviewer #2:

There are some major issues to adress:

1. It should be clarified in the title as well as the abstract that the focus of this paper is on patients with type 2 diabetes on OADs not patients with GLP-1 receptor agonists or insulin.

Responses: Thank you for the review. We have adjusted the title to The impact of ramadan fasting on glucose variability in type 2 diabetes mellitus patients on oral antidiabetic agents. We have also adjusted the abstract.

2. Why were only 3 non-consecutive days of food consumption and activity recorded?

Responses: Thank you for the review. As we know, for the best results we should do food consumption and activity record for seven days. But in systematic review by Pendergast FJ et al 2017 which compare a several ways to do food record and showed that 3-days food record was moderate or good correlations for total energy intake. Thus, we included this issue in the limitation section.

Besides, we used 3 non-consecutive days for food consumption and activity record instead of 7 days record.

3. How would the authors explain the significantly higher intake of fibre at iftar compared to suhur?

Responses: Thank you for the review. There are no studies which explain that higher intake of fiber at iftar than suhur in Ramadan. But, we assumed the higher intake of fiber at iftar because of the culture that Indonesian consume more fruits at iftar.

4. The patients chosen for this study are low to moderate risk and had a relatively high HbA1c level. They spent the majority of the time during CGMS above the interstitial target glucose. The safety of Ramadan fasting measured by glucose variability was shown for this specific group. Studies to confirm the safety of Ramadan fasting for type 2 diabetes subjects with a lower HbA1c or more time in the interstitial target glucose range are still necessary.

Responses: Thank you for the review. Yes, we agree on this point of view.

5. The small sample size is making it difficult to categorize subgroups but differences due to gender would be interesting.

Responses: Thank you for the review. We have added this information in the article.

As well as some minor issues:

1. It should be specified whether the informed consent was attained in written or in verbal form.

Responses: Thank you for the review. Yes, the subject gave written informed consent. We have revised to state that in the articles

2. In the introduction line 66 CGMS is described as a minimally invasive blood glucose examination, whereas in the following sentence it is clearly stated it measures interstitial glucose levels which correlate with blood glucose levels. Also "interstitial glucose" is unnecessarily repeated in brackets.

Responses: Thank you for the review. We have adjusted the sentence.

3. Information on the time when the patients took the medication (morning and/or evening) and daily dosage should be added.

Responses: Thank you for the review. We have added this information in this article. (page 7 line 182-184)

Subjects who were given metformin consumed 500 mg at suhur time and 1000 mg at iftar time. Meanwhile, subjects who were given SU as combination consumed only at iftar time.

Submitted filename: Response to Reviewers.docx

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27 May 2020

The impact of ramadan fasting on glucose variability in type 2 diabetes mellitus patients on oral anti diabetic agents

PONE-D-20-05586R1

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Reviewer #1: Thank you for your changes. Only a little note on my part, please compare the p-value in line 187 with the p-value in table 2 - there is a small discrepancy.

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1 Jun 2020

PONE-D-20-05586R1

The impact of ramadan fasting on glucose variability in type 2 diabetes mellitus patients on oral anti diabetic agents

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