Study protocol for a 15-week randomised controlled trial assessing the independent effects of high-cholesterol and high-saturated fat diets on LDL cholesterol

Abstract

Introduction

Previous research has associated high dietary cholesterol intake with raised low-density lipoprotein cholesterol (LDL-C) and thus increased risk for cardiovascular disease (CVD). Emerging research suggests that it is saturated fat, not dietary cholesterol, associated with increased CVD risk. Despite being high in cholesterol, eggs, low in saturated fat, are not adversely associated with blood lipids or CVD risk. This paper describes a randomised controlled counter-balanced, cross-over trial assessing the effects of a high-cholesterol/low-saturated fat (egg) diet and a low-cholesterol/high-saturated fat diet (egg free) on blood lipids and lipoproteins, while accounting for physical activity levels which can also influence these parameters. The primary aim is to demonstrate that high cholesterol intake (from eggs) within a healthy, low-saturated fat diet does not adversely affect blood lipid levels and lipoprotein profiles. Instead, we propose that adverse effects on these parameters are mediated by saturated fat intake. The secondary aim is to explore relationships between changes in blood lutein and zeaxanthin concentrations and alterations in physical activity, examining whether changes in physical activity mediate effects on blood lipids and lipoproteins.

Methods and analysis

Fifty-two adults aged 18–60 years with LDL-C less than 3.5 mmol/L will be randomly allocated to three isocaloric diets for 5 weeks each: a high-cholesterol (600 mg)/low-saturated fat (6%) (egg) diet, a low-cholesterol (300 mg)/high-saturated fat (12%) (egg free) diet and a control diet that is high in both cholesterol (600 mg) and saturated fat (12%). Lipid and lipoprotein levels, lipoprotein size and concentrations, blood pressure, blood glucose, physical activity levels, and plasma lutein and zeaxanthin concentrations will be measured. Treatment effects will be analysed using linear mixed effects models.

Ethics and dissemination

Ethics approval was obtained from the University of South Australia Human Research Ethics Committee no. 204 327. Results will be disseminated through peer-reviewed journals and national and international presentations.

Trial registration number

NCT05267522

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• Implements highly prescriptive diets to control intake of dietary fat and cholesterol.

• Directly compares the effects of eggs and their contribution to cholesterol and saturated fat intake on lipid and lipoprotein parameters, including particle size and concentration.

• Integrates objective measures of physical activity (accelerometry) to explore the potential causative relationship between increased plasma lutein and zeaxanthin (from eggs) and elevated physical activity, and any moderating effect of physical activity on blood lipids.

• The use of prescriptive diets may not fully replicate real-world dietary patterns.

• The study is reliant on self-reported dietary adherence.

Introduction

Controversy surrounds the ongoing debate over the justification for reducing saturated fat intake, emphasising the need for continued research to understand the complex role of saturated fats in modifying lipids and lipoproteins and therefore cardiovascular disease (CVD) risk.¹ The upcoming study aims to contribute valuable insights into the effects of saturated fat and dietary cholesterol on low-density lipoprotein cholesterol (LDL-C).

Early research indicating that high blood cholesterol levels are associated with an increased risk of developing CVD led to recommendations to limit foods containing high amounts of cholesterol.^2–4 However, most foods with high levels of cholesterol also contain high levels of saturated fat, and emerging evidence suggests that the saturated fat content, rather than cholesterol, increases blood cholesterol levels.^{5 6} Eggs are a somewhat unique food in that while they are rich in cholesterol, they contain little saturated fat.

Data from cross-sectional and prospective studies suggest egg intake does not adversely affect blood lipids or increase CVD risk.^7–12 In fact, some studies support that egg consumption may reduce CVD risk.^13–15 Several randomised controlled trials (RCTs) suggest that eggs do not adversely affect blood lipids^16–19 and may improve high-density lipoprotein cholesterol (HDL-C),^20–24 thus reducing CVD risk. Eggs are rich in choline, which may exert beneficial effects on lipid metabolism.²⁵ Additionally, preliminary evidence suggests lutein and/or zeaxanthin, carotenoids that are present in high concentrations in egg yolk, can increase physical activity by crossing the blood–brain barrier and altering neuronal function,^{26 27} and physical activity may also increase HDL-C.²⁸ Therefore, evaluations of egg consumption on blood lipids should include analysis of lutein/zeaxanthin and physical activity.

Given the growing evidence that cholesterol and saturated fat may exert independent effects on blood lipids, it is important to isolate the contribution of these dietary components, and the foods that contain them, to changes in the blood lipid profile, and thus CVD risk. To date, there has been no direct comparison of the effects of a low saturated fat, high-cholesterol (egg) diet and a high saturated fat, low cholesterol (egg free) diet with a high saturated fat, high-cholesterol (control) diet to determine the independent effects of cholesterol and saturated fat on blood lipids and lipoprotein parameters (particle size and concentration). In addition, no studies of egg intake have assessed sensitive objective markers of physical activity as a potential moderator of effects on blood lipids. This is important given that the lutein and zeaxanthin content of eggs may increase physical activity.

Objectives

Primary objectives

The primary aim of this study is to provide high-quality evidence that a high cholesterol intake, in the context of a healthy low-saturated fat diet, does not adversely affect the blood lipid levels and lipoprotein profile, but rather, that adverse effects on blood lipid levels and lipoproteins are mediated by saturated fat intake. Therefore, we hypothesise that (1) compared with a high-saturated fat, high-cholesterol (control) diet a low-saturated fat, high-cholesterol (egg) diet will result in improved blood lipid levels; (2) compared with a high-saturated fat, high-cholesterol (control) diet a high-saturated fat, low-cholesterol (egg free) diet will result in no difference in blood lipid and lipoprotein levels and (3) a low-saturated fat, high-cholesterol (egg) diet compared with a high-saturated fat, low-cholesterol (egg free) diet will result in improved blood lipid levels.

Secondary objectives

A secondary aim of this study is to investigate potential relationships between changes in blood concentrations of lutein and zeaxanthin and changes in physical activity, and whether alterations in physical activity mediate, at least in part, effects on blood lipids and lipoproteins. Participants will not be made aware of this latter aim as this might influence their physical activity patterns and introduce bias. Participants will only be made aware that plasma lutein and zeaxanthin are being measured as a marker of compliance with egg consumption and that physical activity is being monitored due to its ability to influence blood lipids so it can be controlled for in analyses to determine the independent effects of the diets on blood lipids.

Methods and analysis

Study design

This study uses a randomised, controlled, counter-balanced, cross-over design to evaluate the effect of a high cholesterol, low saturated fat (egg) diet and a low cholesterol high saturated fat (egg free) diet, compared with a high cholesterol high saturated fat (control) diet on the blood lipid profile of healthy adults. Participants will consume three isocaloric diets, in random order, for 5 weeks each:

1. Control diet: high cholesterol diet+high saturated fat.

2. Egg diet: high cholesterol+low saturated fat diet.

3. Egg-free diet: low cholesterol+high saturated fat diet.

Five weeks is sufficient for dietary responses to occur and not require a wash out (ie, this duration of intervention will ensure that previous dietary changes are overridden such that there are no carry-over effects. This approach has been successfully implemented in other studies investigating the effects of diets containing different levels of saturated fat and cholesterol on lipids and lipoproteins.²⁹

If participants cannot complete all three diet phases sequentially, they may pause and continue at a later stage if they remain weight stable (within 2 kg) from their baseline weight. The pause may be up to 5 weeks in duration. The study will be conducted from March 2022 to January 2024 in the clinical trials facilities of the Alliance for Research in Exercise, Nutrition and Activity at the University of South Australia, Adelaide. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines were used in the development of this protocol.³⁰

Patient and public involvement

Participants were not involved in the development of this research protocol. However, all participants will receive a copy of their own personal results on completion, and a copy of the final study results when available.

Participants

Eligibility criteria

Participants will be male and female non-smokers, aged 18–60 years with a blood LDL-C concentration of less than 3.5 mmol/L. Detailed inclusion, exclusion and withdrawal criteria are listed in box 1.

Box 1. Eligibility criteria.

Inclusion criteria

Males and females, aged 18–60 years.

Blood low-density lipoprotein cholesterol <3.5 mmol/L (measured at screening using an automated analyser; Cholestech LDX System).

Non-smoker (minimum 6 months).

Exclusion criteria

Cardiovascular disease.

Type 1 or type 2 diabetes.

Thyroid disorders.

Kidney or liver disease.

Gastrointestinal disorders requiring medical nutrition therapy (eg, Crohn’s disease, irritable bowel, coeliac disease).

Are pregnant or breast feeding.

Have allergies to eggs.

Consume more than 5 eggs per week in the month prior to beginning the trial.

Alcohol (>14 standard drinks/week) (1 standard drink=100 mL wine, 285 mL beer, 30 mL spirit) or drug dependency.

Have changed medication or supplementation that might affect study outcomes in the last 3 months.

Take vitamin, mineral, herbal supplementation or medications that may have an impact on study outcomes.

Are already involved in another research project within 30 days of commencement of the present study that in the opinion of the investigators will be unsuitable for this study.

Show unwillingness to follow any of the diets.

Failure to satisfy the investigator regarding suitability to participate for any other reason.

Are unwilling or unable to provide written consent.

Withdrawal criteria

Adverse reaction to diets.

The need to take a medication or treatment, which in the opinion of the investigator, may interfere with study measurements.

Weight loss or gain more than 5% of their baseline body weight.

Withdrawal of consent by the participant.

Failure to satisfy the investigator regarding suitability to participate for any other reason.

Recruitment/screening

Participants will be recruited from the public using advertisements on social media platforms and online forums. Prospective participants will be directed to an online database Research Electronic Data Capture (REDCap) to view the participant information sheet and consent form (PICF), which outlines the requirements of the study and the need for informed consent, and contains a diet and lifestyle questionnaire (DLQ) that participants need to complete to determine their eligibility to participate in the study. Alternatively, they may request to receive a hard copy of the PICF and DLQ via post or email. After reviewing the DLQ responses, eligible participants will attend a screening appointment to review their medical history, concomitant medication and supplementation, and measure their LDL-C. Participants who meet the required criteria will be invited to provide written informed consent (online supplemental file 1) in the presence of the investigator and invited to attend a baseline appointment at least 8 days later.

Randomisation, allocation concealment and sequence generation

Participants will consume three isocaloric diets, in random order, for 5 weeks each. The order in which the diets are consumed will be randomised and balanced. The randomisation sequence generation incorporated six variations (ABC, ACB, BCA, BAC, CAB, CBA), ensuring a balanced allocation of participants to the different diet orders. Participants were assigned to one of these sequences, and they followed the corresponding diet order throughout the study. This design was chosen to minimise the potential impact of order effects. Randomisation to each starting diet will be via minimisation,³¹ based on the LDL-C concentration at screening and sex. Minimisation will ensure balanced LDL-C and sex between the treatment groups at commencement of each diet and has been proposed to be the best randomisation method for small clinical trials to reduce bias.^{31 32} The treatment allocation will be performed by a staff member who is independent of the study outcome assessments. The randomisation list will be maintained in a secure location with access only to authorised personnel. Participants cannot be blinded to the diets they consume, nor can the research dietitian who assists them in complying with the diets. The researchers conducting outcome assessments and statistical analysis will remain blinded until the completion of statistical analysis.

Sample size calculation

The study is powered on the primary outcome LDL-C. It is hypothesised that compared with the two high saturated fat diets (control and egg free diets), the egg diet, which is low in saturated fat will result in significantly lower LDL-C. An earlier intervention evaluating the effect of differing intakes of beef on blood lipids (diets similar in saturated fat content to the present study) reported a difference in LDL-C of 0.21±0.1 mmol/L between high and low saturated fat diets.²⁹ Anticipating a similar effect in the present study, a sample size of 42 participants will provide 80% power to detect a difference in LDL-C of 0.21±0.1 mmol/L between diets (α-level of 0.05). To account for approximately 20% drop-out, at least 52 participants will be recruited to allow the required 42 participants to complete.

Preintervention

During the week prior to the baseline visit, the sleep patterns and physical activity levels of participants will be monitored using a wrist-worn accelerometer (Axivity AX3) for 7 days. They will also complete a 5-day weighed food diary (non-consecutive days with 1-weekend day). See table 1 for all outcome measures at each time point. The baseline assessment will be scheduled a minimum of 8 days after the screening appointment. Participants will be asked to refrain from alcohol for 24 hours and fast for 10–12 hours prior to each assessment in preparation for blood sampling. At the baseline assessment, they will return the accelerometer and the diaries provided at screening. This process will be repeated at the start of each diet phase (weeks 5 and 10) and the accelerometer and final diaries will be returned at the end of the trial (week 15).

Table 1.

Outcome measures at each time point

	Screening	Prebaseline (enrolment)	Baseline (allocation)	Diet 1					Diet 2					Diet 3
Time point (weeks)	−3	−1	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Anthropometry
Height			X					X					X					X
Weight (and body mass index)			X					X					X					X
At-home Bluetooth scale weight				X	X		X		X	X		X		X	X		X
Waist circumference			X					X					X					X
Blood pressure
Seated blood pressure (SBP/DBP)			X					X					X					X
Physical activity and sleep
Accelerometry to measure sedentary, light and moderate-vigorous physical activity		X					X					X					X
Sleep diary to measure sleep patterns		X					X					X					X
Dietary intake
5-day food diary (paper or Easy Diet app) to assess energy and macro/micronutrient intake		X					X					X					X
Daily food checklist to monitor dietary compliance				X	X	X	X	X	X	X	X	X	X	X	X	X	X	X
Blood sample analysis
LDL-C screening	X
Fasting lipids and lipoproteins
Triglycerides and subclasses (concentration and particle size)			X					X					X					X
TC, LDL-C, HDL-C, IDL-C, VLDL-C and their subclasses (concentration and particle size)			X					X					X					X
Apolipoprotein B			X					X					X					X
Apolipoprotein A1			X					X					X					X
Glucose			X					X					X					X
Biomarkers of compliance
Plasma lutein and zeaxanthin			X					X					X					X

DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; IDL-C, intermediate-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol; VLDL-C, very low-density lipoprotein cholesterol.

Study intervention

Participants will consume three isocaloric diets, in random order, for 5 weeks each:

1. Control diet (average Australian diet): high cholesterol diet+high saturated fat (approximately 35% fat (12% saturated fat), 25% protein, 40% carbohydrate, 600 mg cholesterol) (one egg per week).

2. Egg diet: high cholesterol+low saturated fat diet (approximately 35% fat (6% saturated fat), 20% protein, 45% carbohydrate, 600 mg cholesterol) (2 eggs/100 g per day).

3. Egg-free diet: low cholesterol+high saturated fat diet (approximately 35% fat (12% saturated fat), 25% protein, 40% carbohydrate, 300 mg cholesterol) (no eggs).

The control and egg-free diets will provide 12% of energy as saturated fat, reflective of the typical Australian diet, with the 2011–2012 Australian National Nutrition and Physical Activity Survey indicating that the average saturated fat intake was 12% of energy.³³ The Australian Dietary Guidelines indicate that the average dietary intake of cholesterol is 600 mg/day.³⁴ Therefore, for the high cholesterol diet we will target a cholesterol intake of 600 mg/day, and for the low cholesterol diet we will target an intake of 300 mg/day. In this way, the control diet will reflect the typical Australian diet (ie, 600 mg/day of cholesterol and 12% saturated fat) and is thus an appropriate control diet. All diets will be matched as closely as possible for protein and carbohydrate. The dietary composition will prominently include complex carbohydrates sourced from whole grains, fruits and vegetables rich in fibre, vitamins and minerals—nutrients typically associated with heart-healthy choices.^{1 35} Additionally, the diets will limit refined carbohydrates and added sugars, recognising their established link to an increased risk of CVD.³⁶ The variation in macronutrients between the diets are a result of deliberate manipulations aimed at achieving desired cholesterol and saturated fat levels. For example, the inclusion of more animal protein in the saturated fat diet increases the overall protein content. Additionally, differences in food selection to control cholesterol and saturated fat levels will lead to slight variations in micronutrient profiles. Alcohol intake will be limited to two standard drinks per week during the study. The diets will be prescribed as specific quantities of food, based on Australian Dietary Guidelines, and individualised for the participant’s energy requirements to achieve weight maintenance. Energy requirements will be determined by using the Schofield equation, based on age, sex and baseline body weight, as well as self-reported physical activity.³⁷ Diets will be presented in a checklist format that will be completed daily and participants will be provided with kitchen scales and measuring cups and spoons to assist in measuring out the appropriate food quantities in line with the food checklists.

After completing baseline assessments, participants will be provided with education and resources from the dietitian for the first randomised diet phase. Each diet phase will include eight detailed meal plans with recipes, which will be used on rotation for the 5-week period. Throughout each dietary phase, participants will attend three diet review consults (via video conference or phone) and receive individualised dietary advice (20–30 min each). The study dietitian will assess dietary compliance and provide strategies to maintain dietary compliance. Body weight will be reviewed to ensure participants maintain their starting weight. The dietitian will give advice to assist with weight maintenance if required. Daily checklists of serves of food eaten will be completed and checked at each dietitian session. Participants will be asked to complete a 5-day weighed food record at baseline and during the final week of each 5-week diet phase. Participants will be asked to record all foods and drinks consumed and to record weights or estimate volumes using standard measures where possible and provide details of branded products. If they do not have their own, participants will be provided with a set of kitchen food scales. Participants will be supplied with financial support to purchase key study foods to the value of $A55 per week ($A825 total). Figure 1 outlines the study timeline.

Figure 1.

Study timeline.

Data collection

The following section outlines the data and biochemical samples being collected during the intervention periods (see table 1 for a summary).

Anthropometry

Anthropometric assessments will be conducted with participants barefoot and wearing light clothing. Height will be measured twice to the nearest 1 mm using a stadiometer (baseline only) (SECA 216 Height Measuring Rod, SECA), and the average recorded. Body weight will be recorded to the nearest 100 g and will be measured twice on each occasion using calibrated electronic scales (SECA 703 Digital Column Scales, SECA). The average value will be calculated, and the same scales will be used throughout the intervention. BMI will be calculated as weight/height squared (kg/m²). Waist circumference will be measured at the narrowest point of the abdomen or, if there is no obvious narrowing, at the midpoint between the lower costal (10th rib) border and the iliac crest.³⁸ Three measurements will be taken, and if they differ by more than 2%, a fourth measurement will be obtained. The mean of the measurements will be used for analysis. In addition, participants will be provided with scales (Withings/Nokia WBS06, Nokia) with Bluetooth capacity to monitor their weight at least twice per week at home and these data will be sent to research staff to assist with weight monitoring.

Blood pressure

Seated blood pressure will be measured in a controlled environment using an automated sphygmomanometer and appropriately sized cuffs after a 5 min quiet rest, following Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure 7 guidelines.³⁹ The same arm will be used for all assessment visits. Three consecutive readings will be taken at approximately 2 min intervals, and the mean of the three measurements will be used for analysis.

Accelerometry for physical activity and sleep monitoring

To assess physical activity during the intervention, accelerometry (Axivity AX3) will be used to ascertain the amount of time spent sedentary and engaged in light or moderate-vigorous physical activity. Participants will wear the accelerometers on their non-dominant wrist for seven consecutive days, including during sleep. The signal vector magnitude of the acceleration, minus gravity, will be computed and summed over 1 min epochs. Raw data (each 1 min epoch) will be extracted using the open-source OMGUI Configuration and Analysis Tool (Axivity, Newcastle, UK). These data will then be imported into custom software (Cobra, developed at the University of South Australia, Adelaide, Australia) through MATLAB R2019a (MathWorks, Natick, Massachusetts, USA). Non-wear will be identified using the method of Choi et al.⁴⁰ Valid days will include at least 10 hours of waking wear time. Data for participants with fewer than four valid days will be excluded. Each 1 min epoch of waking wear time will be classified as either sedentary, light, moderate or vigorous physical activity using Esliger’s cut points for adults.⁴¹ Participants will be provided with a paper-based record sheet to document sleep patterns and non-wear time: (1) the time they went to bed (‘bedtime’), (2) the time they woke up (‘get up time’) and (3) the time the device was removed (‘non-wear’) and put back on again including the reason for removal (eg, showering).

Biochemical measures

LDL-C screening

Participant’s finger prick blood will be screened for LDL-C using an automated analyser (Cholestech LDX System, Abbott) with CardioChek blood collection tubes, calibration solutions and test strips.

Lipids and lipoproteins

Fasting venous blood samples will be collected (approximately 15 mL) and all samples centrifuged (4°C, 4000 rpm, 10 min) to separate plasma and stored at −80°C for later analysis. Plasma (EDTA) samples will be analysed by LabCorp (LipoScience, Morrisville, North Carolina, USA) who will provide a standard clinical lipid panel (triglyceride, total cholesterol, HDL-C, LDL-C, very low LDL-C, non-HDL-C) and apolipoprotein (ApoB, ApoA-1) concentrations. LDL-C is calculated via the National Institutes of Health equation.⁴² Particle concentrations of triglyceride-rich lipoproteins, LDL-C and HDL-C subclasses will be assessed by nuclear MR (NMR; LipoScience, Morrisville, North Carolina, USA). The analysis includes concentrations (nmol/L, μmol/L) and sizes (nm) of lipoprotein particles.⁴³ For a full list of lipid analyses, see table 2.

Table 2.

Lipid and lipoprotein parameters that will be assessed

Parameter
TRLP concentrations (nmol/L)	Total-TRLP, VL-TRLP, L-TRLP, M-TRLP, S-TRLP, VS-TRLP
cLDLP concentrations (nmol/L)	Total-cLDLP, L-cLDLP, M-cLDLP, S-cLDLP
cHDL concentrations (µmol/L)	Total-cHDLP, L-cHDLP, M-cHDLP, S-cHDLP, H7P, H6P, H5P, H4P, H2P, H1P
Mean particle sizes (nm)	TRLZ, LDLZ, HDLZ
Lipid concentrations (mg/dL)	TRL-TG, TRL-C
Extended lipid panel (mg/dL)	TG, TC, HDL-C, LDL-C, VLDL-C, Non HDL-C
Apolipoproteins (mg/dL)	ApoB, ApoA-1

ApoA-1, apolipoprotein A-1; ApoB, apolipoprotein B; cHDLP, calibrated high-density lipoprotein particle; cLDLP, calibrated low-density lipoprotein particle ; HDL-C, high-density lipoprotein cholesterol; HDLZ, mean high-density lipoprotein particle size; H7P-H1P, HDLP subspecies; L, large; LDL-C, low-density lipoprotein cholesterol; LDLZ, mean low-density lipoprotein particle size; M, medium; NMR, nuclear MR; S, small; TC, total cholesterol; TG, triglyceride; TRL-C, triglyceride-rich lipoprotein cholesterol; TRLP, triglyceride-rich lipoprotein particle; TRL-TG, triglyceride-rich lipoprotein triglycerides; TRLZ, mean triglyceride-rich lipoprotein particle size; VL, very large; VLDL-C, very low density lipoprotein cholesterol; vs, very small.

Glucose

Fasting plasma (potassium oxalate/sodium fluoride) glucose will be measured using a Konelab auto analyser (%CV<5%).

Biomarkers of compliance

Given that egg yolk is a major source of the carotenoids lutein and zeaxanthin and consumption of one egg per day has been shown to significantly increase circulating concentrations of these carotenoids,⁴⁴ plasma (EDTA) lutein and zeaxanthin will be measured as a marker of compliance with egg consumption. Plasma samples will be stored at −80°C until the end of the study and then lutein and zeaxanthin will be determined according to the method of Wood et al.⁴⁵

Dietary analysis

Adherence to diets will be assessed by analysing 5-day weighed food records using Foodworks Nutritional Analysis Software version 10 (Xyris Software, Brisbane, QLD, Australia). This will provide an estimate of daily energy, macronutrient and micronutrient intake.

Data management

Each participant will be allocated a unique code to identify their data and biological samples. Personal identifiers (name, email address and phone number) will be stored in a password-protected computer database accessible only by the researchers. Data will be entered into a REDCap database at all clinic and online visits. All data will be securely stored at the University of South Australia for 15 years after which it will be destroyed. Biological samples will be stored at −80°C in a secure freezer facility in temperature monitored freezers. Samples will be analysed in a single batch at the end of the study to reduce the impact of interassay variability.

Protocol deviations

Any deviations from the proposed protocol will be communicated via an update of the ClinicalTrials.gov registration and through a letter to the editor of this journal.

Adverse events

Any adverse events that occur during the study will be recorded in REDCap and reported to the University of South Australia Human Research Ethics Committee. Adverse events leading to participant withdrawal will be reported in future publications. There are no plans to conduct a formal analysis of adverse events.

Statistical analysis plan

Statistical analysis will be performed using Stata/IC V.18 (StataCorp). We will test for order effects to check for no carry over effects. The effects of the different diets on the dependent measures will be analysed using linear mixed effects models, with diet treatments being fixed effects and participant ID a random effect. If participants drop out, provided the data are missing at random, all available data will be used in the analysis. Thus, the analysis performed will constitute an intention-to-treat analysis. A sensitivity analysis will also be performed using only data from participants who completed all aspects of the protocol (ie, no missing data). Linear mixed effects models will also be used to assess relationships between lutein and physical activity. Statistical significance will be set at an α-level of 0.05.

Data access

There are no contractual agreements that require the data from this trial to be shared.

Ethics and dissemination

Ethics approval was obtained from the University of South Australia Human Research Ethics Committee (204327).

Participants will receive a copy of their individual results as well as a summary of the study findings. Participants who complete all aspects of the study will receive an honorarium of $A600 to compensate for their time and travel expenses. This is in addition to the financial support to purchase key study foods. Participants who withdraw from the study will be provided with a pro rata of the $A600 reimbursement for participation. The study’s findings will be shared through scientific conferences and published papers. In addition, relevant outcomes will be made accessible to the public through media releases as deemed appropriate.

Discussion

For decades, dietary cholesterol was implicated in increasing LDL-C levels leading to development of CVD.^2–4 However, foods that are rich in cholesterol are also typically high in saturated fat, and recent evidence suggests that it is the saturated fat rather than cholesterol that is associated with an increased risk of CVD.^{5 6} Eggs are high in cholesterol but low in saturated fat and while there is some evidence that egg consumption does not increase the risk of CVD or negatively affect blood lipids,^7–12 high-quality RCTs are required to confirm this. Furthermore, eggs may even reduce the risk of CVD^13–15 and improve HDL-C levels.^20–24 Choline, which is abundant in eggs, may help regulate lipid metabolism and thus influence CVD risk.²⁵ Lutein and zeaxanthin in egg yolk may also increase physical activity,^{26 27} which can in turn improve HDL-C levels. Therefore, further investigation is required to evaluate the effect of egg consumption on blood lipids and physical activity to better understand the impact of cholesterol and saturated fat on blood lipids and CVD risk using rigorous, high-quality, RCTs.

Strengths

The randomised-controlled design of this study will yield high-quality evidence demonstrating that a low-saturated fat, high-cholesterol diet with eggs does not negatively affect LDL-C or other blood lipids compared with a low-cholesterol, high-saturated fat diet without eggs. The study will highlight the role of saturated fat intake in mediating adverse lipid and lipoprotein effects. The study will also assess novel lipoprotein parameters, such as particle size and concentration, to advance understanding of how dietary cholesterol affects blood lipids. Additionally, this study incorporates several innovative elements. It will be the first trial to explore the effect of increased plasma lutein and zeaxanthin from egg consumption on physical activity and determine whether any changes in physical activity may affect blood lipids. This information will provide preliminary evidence for future studies to explore the potential of eggs for improving a range of health outcomes that are influenced by physical activity.

Limitations

One of the main challenges with any dietary intervention study is recruiting participants and keeping them motivated and compliant with the required dietary modifications. This study requires participants to follow a strict diet for 15 weeks. We have previously successfully recruited participants for similar intervention trials and plan to support participants with regular appointments with a dietitian, as well as the provision of simple food checklists and financial assistance to cover the costs of purchasing the necessary foods to assist with compliance.

Ultimately, a positive outcome from this 15-week randomised controlled cross-over trial will add to the evidence base on dietary fat and cholesterol intake and lipid and lipoproteins, encouraging healthcare professionals to recommend eggs as a nutrient-dense protein source as part of a healthy diet.

Ethics statements

Patient consent for publication

Not applicable.