Acute Evidence of Digestive, Metabolic and Nutritional Differences in Beef and Meat- Analogue Meals
Phase: NA
Conditions: Diet, Healthy, Protein; Disease
Sponsor: University of Auckland, New Zealand

1 
 
TITLE 
Acute Evidence of Digestive, Metabolic and Nutritional Differences in Beef, Lamb and Meat-
Analogue Meals: A study protocol for a randomised control trial 
 
Names protocol contributors: Andrea Braakhuis1, David Cameron-Smith2, Toan Pham1, 
Scott Knowles3, Matthew Barnett3,4, Lovedeep Kaur4,5, Emma Bermingham3 
1The University of Auckland, Faculty of Medical and Health Sciences, Grafton Campus, 
Auckland, New Zealand; a.braakhuis@auckland.ac.nz; toan.pham@auckland.ac.nz 
2Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, 
Singapore; DCameron_Smith@sics.a-star.edu.sg 
3Food & Bio-based Products Group, AgResearch Grasslands, Palmerston North 4442, New 
Zealand; 
scott.knowles@agresearch.co.nz; 
matthew.barnett@agresearch.co.nz; 
emma.bermingham@agresearch.co.nz 
4Riddet Institute, Palmerston North 4442, New Zealand 
5School of Food and Advanced Technology, Massey University, Palmerston North 4442, New 
Zealand; l.kaur@massey.ac.nz 
 
Corresponding author: A. Braakhuis; a.braakhuis@auckland.ac.nz 
 
 

2 
ABSTRACT 
Background 
Plant-based analogues and substitutes for animal meat are receiving considerable attention 
within academia and the popular press. Once a niche product aimed at vegetarians, they are 
increasingly promoted to omnivores and meat-moderating “flexitarians”. Proponents view 
these new foods as a means of reducing livestock production and improving the environment, 
whilst industry sees opportunity to repurpose traditional and novel crops into high-value 
ingredients. To date, most research on meat alternatives has focused on public perception, 
processing technologies, hedonics and the consumer acceptability issues that underpin product 
development. There have been surprisingly few investigations into the nutritional, health and 
well-being consequences of consuming meat analogues over the short-term or long-term. The 
aim of our current trial is to investigate the digestion, absorption and utilisation of a meat-
analogue meal in Millennial consumers and compare that to matched meals featuring pasture-
raised, grain-finished beef or lamb. 
Methods 
A cohort of healthy, young (20-34 years old) male participants will consume a single meal on 
four occasions in a treatment-crossover, blinded investigation. Each instance will be followed 
by four hours of repeated venous blood collection to assess the digestive and metabolic effects 
of the meal. The four meals include a popular commercial meat analogue comprising pea, bean 
and rice extracts plus plant fats and cellulose, or a mince from pasture-raised or grain-fed beef 
or grass-raised lamb. Blood plasma samples of the participants will be measured for changes 
in chylomicron fatty acid distribution, amino acid profile, neurotransmitter proteins, minerals, 
inflammatory markers and biomarkers of general health. 
Discussion 
To our knowledge this is the first randomised controlled trial (RCT) comparing the acute 
nutritional effects of iso-caloric, blinded meals containing a plant-based meat analogue or 
pasture-fed-, grain-finished beef or lamb. The study provides proof of principle for only the 
effects of a single meal. Future work will translate these insights into longer-term, lifestyle 
interventions. 
Trial Registration 
This study, registered as Universal trial number: U1111-1244-9426,  will be carried 
out under the auspices of The University of Auckland. The trial has been granted 
ethical approval by the Ministry of Health, Health and Disability Ethics Committee (Ref: 
19/STH/226).  
Date and version: 24/07/20, 5 
Key Words: protein, amino acid, diet, health, meat products, bioavailability 

3 
 
ADMINISTRATIVE INFORMATION 
Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. 
The order of the items has been modified to group similar items (see http://www.equator-
network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-
clinical-trials/). 
Table 1: Trial Administrative Details 
Title {1} 
Acute Evidence of Digestive, Metabolic and Nutritional Differences in Beef, Lamb 
and Meat-Analogue Meals: A study protocol for a randomised control trial 
Trial registration {2a 
and 2b}. 
Universal trial number: U1111-1244-9426. ClinicalTrials.gov: 5000927, 
(pre-recruitment) 
Protocol version {3} 
24/07/2020, Version 5 
Funding {4} 
This research is funded by the Meat Industry Association Innovation Limited (a 
subsidiary of the New Zealand Meat Industry Association), Beef and Lamb New 
Zealand Limited and the New Zealand Ministry of Business, Innovation and 
Employment.  
Author details {5a} 
Refer to Table 3 
Name 
and 
contact 
information for the trial 
sponsor {5b} 
Auckland Uniservices, c/o The University of Auckland, Private Bag 92019, 
Auckland 1142, New Zealand 
Role of funder{5c} 
The funder(s) have no involvement in the data collection, analysis and 
interpretation of the data; in the writing of the report; and in the decision to submit 
the paper for publication as contracted. 
 
INTRODUCTION 
Background and Rationale 
A popular and common dietary trend is the reduction in red meat, and increase in meat 
alternatives. Whilst meat alternatives were once niche food products aimed at vegetarians, they 
are increasingly marketed to omnivores and “flexitarians”, thus contributing to a trend for 
reductions in red meat intakes [1]. While the environmental effects, consumer perceptions and 
acceptability of meat alternatives have been investigated [2, 3, 4], yet there is surprisingly a 
paucity of data comparing the nutritional and digestive differences to meat.  
Red meat consumption contributes essential nutrients to the diet, including balanced amino 
acids, long chain fatty acids and complex lipids, vitamins (including B12) and minerals 
(including iron, zinc and selenium) [5]. Such nourishment comes in the context of a whole food 
that also includes health risk components, such as endogenous saturated fats, nitrates added to 
preserved meats, or heterocyclic amines formed during cooking [5]. Consequently, 
categorising a complex food as being either ‘good’ or ‘bad’ for health is now considered overly 
simplistic [6].  

4 
 
Dietary substitutes for red meat have a long history and new options are being actively 
developed. Most plant-based analogues attempt to replicate the taste and eating experience of 
meat, whilst delivering quality nutrition. The products’ nutrition information panels (NIP) and 
label claims emphasise chemical analysis (primarily amino acid profiles) and draw 
comparisons to the acknowledged standard of muscle meat. This composition is indicative of 
potential nutrition, but it is not definitive, particularly for highly processed foods. The matrix 
of a food, its ingredients and structure, have marked effects on how well nutrients are actually 
absorbed and utilised. Few research studies have addressed the health outcomes of consuming 
novel meat analogues or compared them directly to traditional foods.  
Red meat is also a complex food that reflects its origin and handling. Differences arise due to 
species, farming and feeding practices of the animals [7]. For instance, meat from ruminant 
animals raised on predominately pasture-based diets has higher levels of polyunsaturated fatty 
acids (PUFA) and related lipids derived from leaf oils (i.e. green grass) compared to meat from 
grain-fed animals, which accumulates the lipids of seed oils [8]. Research on the clinical 
implications of consuming these different fatty acid profiles is contradictory and may reflect 
health status and habitual patterns of meat consumption. Nevertheless in healthy consumers, 
pasture-raised meat increased both plasma and platelet concentrations of long-chain n-3 PUFA 
(LCPUFA) [9], supporting the effects of meat-derived fatty acids on health indices [10].  
The LCPUFA subset found in red meat is drawing increasing attention as important bioactives 
for health, given their lipid-lowering properties, reductions in platelet aggregation, 
inflammation, and improvements in cognition and mood [11]. In the context of a Western diet 
where seafoods are minimal, the contribution of red meat to total LCPUFA intake can be 
substantial [12]. Research has shown that the LCPUFA of pasture-raised Wagyu was twice that 
of grain-finished Angus [8] and that it would contribute approximately 35-80% to the 
recommended adequate intake for men and women, respectively [13]. 
Meat is highly digestible and its nutrients are readily available. The qualities of grass- and 
grain-finished beef have not been directly compared to a plant-based meat analogue and lamb, 
outside of glycaemic and satiating responses [14]. Our proposed research will provide insight 
into the digestibility and nutrient delivery of these foods, and will quantify key metabolic 
responses to consuming a single meal. This study is a step towards understanding the 
nutritional, health and well-being consequences of meat analogues that may accrue from long-
term repeated consumption.  
Objectives 
To investigate the digestive responses to an acute intake of pasture-fed beef, grain-finished 
beef, lamb and a plant-based meat analogue consumed as a component of a mixed meal.  
This study is part of a larger programme to understand the nutritional implications of 
consuming NZ, pasture-fed red meat as part of a balanced diet. 
Trial Design 
A 4-arm randomised control trial using crossover design with equal allocation to each arm and 
adopting a non-inferiority framework. 
 

5 
 
METHODS/DESIGN 
Study Setting 
The trial will be conducted at the University of Auckland Clinical Research Centre. The study 
is a randomised crossover trial to capture biological difference in postprandial nutrient 
dynamics. Research subjects will act as their own controls and will consume each meal on a 
separate occasion in random order. The study compares exemplars of pasture-fed New Zealand 
beef, grain-finished New Zealand beef, New Zealand lamb and a plant-based meat analogue. 
The pasture-fed beef was obtained from Angus Steers that had been fed exclusively on pastures 
(ryegrass and white clover mix) whereas the grain-finished beef was Angus steers that had been 
finished for an average of 122 days on a maize silage, barley wheat and straw ration. Marble 
score (3-4/9 on the Australian Grading System) [15] and pH (average 5.5) of the meat were 
consistent between production systems. The lamb will be sourced from New Zealand. The meat 
analogue has been selected from commercial varieties on the basis of its macronutrient profile 
(protein and fat) and appearance to best match that of meat.  
The digestion and metabolism of key nutrients will be measured immediately after the ingestion 
of a single meal. This experimental setting will also be used to examine some subjective 
qualities of the meal experience, such as satisfaction score (i.e., liking, satisfaction, enjoyment, 
satiety, appetite) and gastrointestinal score (i.e., comfort, fullness, bloating, rumbling, 
flatulence, faecal urgency, diarrhoea). 
Eligibility criteria 
All participants will be omnivores willing to consume both red meat and plant-base alternatives 
for the purposes of the trial. Those with chronic health conditions, hyperlipidaemia, obesity 
(BMI ≥ 30), use of medications (except occasional use of NSAIDs and antihistamines), history 
of anosmia and ageusia (issues with taste and smell), current dieting or disordered eating 
pattern and smoking tobacco or recreational drugs will be excluded from participating. 
Participants will complete an on-line screening that includes the Three-factor Eating 
Questionnaire-R18 (TFEQ) and a health survey. Participants with a TFEQ score greater than 
75% will be excluded from participating on the basis their perception of food is potentially 
influenced by underlying psychological issues.  
Recruitment and Informed Consent 
Males will be recruited from the millennial generation (20-34 y), as males typically have a 
greater postprandial lipid response than females [16]. In general, Millennials are chosen as this 
population demographic has been demonstrated to have the greatest variation in meat intake 
[17]. Recruitment will occur via posters placed around the University of Auckland and using 
social media sites (FacebookTM). Informed consent will be collected by research staff following 
participant’s inquiry and provision of information. 
Ethics and dissemination  
The trial has been granted ethical approval by the New Zealand Ministry of Health’s Health 
and Disability Ethics Committees (Ref: 19/STH/226). All results originating from this study 
will be submitted for publication in scientific journals and presented at meetings. 
Sample size calculation 

6 
 
The principal biomarker for calculation of sample size is postprandial change in LCPUFA 
concentrations in plasma chylomicrons (namely 20:4 n-6, 20:5 n-3, 22:5 n-3, 22:6 n-3). We 
estimate a sample size of 29 would enable the detection of a smallest worthwhile change of 0.5 
mmol/L from baseline to 4 hours post meal consumption [19]. Pooled published data indicate 
an increase of ~3.9 mmol/L (±2.8 mmol/L) in chylomicron fatty acids post meal consumption. 
An effect size of 0.2 is used to calculate the smallest worthwhile effect. Recruitment will 
continue until 29 men have successfully completed the trial. 
Randomisation and Blinding 
The design will be a single blinded crossover. Participants will consume each test meal in 
random order, using a computer-generated sequence, at least one week, but no more than one 
month, apart. The randomisation will be conducted centrally, with one research staff 
responsible for meal preparation aware of the allocation. Staff responsible for blood collection 
and analysis will be blinded to the intervention, as will participants. 
Reminder text messages will be sent to participants to increase attendance to each of the four 
study appointments. Additionally, participants will receive financial compensation for 
partaking in the end of four assessment visits. Participants will have the right to withdraw from 
the study at any time. The principal investigator will have the right to discontinue participants’ 
involvement in the study if they become ineligible or when significant protocol deviations 
occur. The data of participants who withdraw will be kept and might be used in exploratory 
analyses, unless the participant requests for the data to be deleted. 
Study Overview 
Two test meals will include the same cut and quality of pasture-fed New Zealand beef or grain-
fed New Zealand beef of well-defined provenance, which has been specifically slaughtered, 
packaged and stored for this trial. The third test meal will be lamb meat that will be sourced 
from New Zealand as a representative sample. The fourth test meal will be a commercial plant-
based meat alternative that best resembles in terms of appearance and macronutrient profiles. 
Meals will include a 220 g raw serving of beef or lamb (approximately 160 g cooked), which 
is in line with the World Cancer Research Fund / International Agency for Research on Cancer 
recommendation on red meat consumption, which suggests limiting the weekly red meat 
consumption to 350-500 g cooked. A minimum quantity of 100 grams cooked meat is required 
to ensure adequate fat intake to allow assessment of post-meal lipid dynamics [19]. The test 
meals will be provided in the morning after an overnight fast. The meals will look identical in 
appearance and participants will not be told which meal they have received.  
Saliva samples will be collected at one time prior to meal consumption (baseline). These 
samples will be sent for subsequent analysis at the Nutrigenomix Inc laboratory. The test 
analyses variations in 70 genes that impact nutrient metabolism, eating habits, weight 
management and body composition, food intolerances and physical activity. The accuracy of 
the genetic test results are between 99.7 – 100%. 
Blood samples will be collected immediately prior to (baseline) and 60, 120, 180 and 240 
minutes following the meal for serum and EDTA plasma. Bloods will be taken in the 
phlebotomy room in the research facility, by a phlebotomist using via an indwelling catheter. 
Blood samples will be processed and frozen at -80°C for subsequent analysis.  

7 
 
During this time of each visit, participants will remain in the research unit. Appetite, digestive 
comfort and palatability scores will be self-reported using manual questionnaires. 
The SPIRIT reporting guidelines for the accurate documentation of clinical trial protocols has 
been followed [16]. See Table 1 for trial schedule. 
Table 2.  SPIRIT Chart. Schedule of enrolment, interventions, and assessments 
 
 
STUDY PERIOD 
 
Enrolment 
Allocation 
 
 
Post-allocation 
Close-
out 
TIMEPOINT 
-t1 
0 
tpre 
t0 
t30 
t60 
t120 
t180 
t240 
+t1 
ENROLMENT: 
 
 
 
 
 
 
 
 
 
 
Eligibility screen 
X 
 
 
 
 
 
 
 
 
 
Informed consent  
X 
 
 
 
 
 
 
 
 
 
Allocation 
 
X 
 
 
 
 
 
 
 
 
INTERVENTION 
MEALS: 
 
 
 
 
 
 
 
 
 
 
Grain-fed Beef 
 
Random 
order 
 
X 
 
 
 
 
 
 
Grass-fed Beef 
 
Random 
order 
 
X 
 
 
 
 
 
 
Lamb 
 
Random 
order 
 
X 
 
 
 
 
 
 
Meat Alternative 
 
Random 
order 
 
X 
 
 
 
 
 
 
ASSESSMENTS: 
 
 
 
 
 
 
 
 
 
 
Amino acids  
 
 
X 
 
 
X 
X 
X 
X 
 
Neurotransmitters 
 
 
X 
 
 
X 
X 
X 
X 
 
Inflammatory 
Markers 
 
 
X 
 
 
X 
X 
X 
X 
 
Chylomicron fatty 
acids 
 
 
X 
 
 
X 
X 
X 
X 
 
Chylomicron 
Lipidomics 
 
 
X 
 
 
 
 
 
X 
 
Glucose/ Insulin 
 
 
X 
 
 
X 
X 
X 
X 
 
Vitamins (ADEK) 
 
 
X 
 
 
X 
X 
 
X 
 
Vitamins (Bs) 
 
 
X 
 
 
X 
X 
 
X 
 
Haemoglobin/ 
Minerals 
 
 
X 
 
 
X 
X 
X 
X 
 

8 
 
Saliva 
genetic 
markers 
 
 
X 
 
 
 
 
 
 
 
Questionnaire-
Fullness 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Questionnaire-
Digestive 
Symptoms 
 
 
X 
X 
X 
X 
X 
X 
X 
 
Questionnaire-
Palatability 
 
 
 
 
X 
 
 
 
 
 
Questionnaire- 
Participant Insight   
 
 
 
 
 
 
 
 
X 
 
Intervention 
All meals will be prepared according to standardised recipes by the Research Dietitians and 
served at the test kitchen site in The University of Auckland Clinical Research Centre. The 
meat will be minced to ensure homogeneity between participants. All non-meat food items will 
be purchased at a local supermarket. The test diets will be protein and fat matched, and the 
meals will be balanced for energy, carbohydrate and fibre. A panel of meat scientists, a 
nutritionist and research dietitian reviewed available options to choose the plant-based meat 
analogue. Their criteria included 1) nutrition should approximate beef, 2) appearance should 
approximate beef, and 3) readily available in the retail space. Nutrient content data for the beef 
mince was based on New Zealand composition tables, and for the analogue was taken from the 
NIP. 
All participants will be fasted for 10 hours prior to consuming the test meal. All test meals will 
be given in the morning, as postprandial lipid responses are greatest at this time [17].  
Outcomes (Primary and Secondary)  
Following the consumption of a single meal: 
Primary outcome variable  
• Change in LCPUFA (18:2 n-6, 18:3 n-3, 20:4 n-6, 20:5 n-3, 22:5 n-3, 22:6 n-3) 
concentrations in the chylomicron fraction over 4 hours. Difference in the change 
between pasture-, grain-beef, lamb and plant-based alternative meat meals. 
Secondary outcome variables  
• Differences in other fatty acid (14:0, 16:0, 16:1 n-7, 18:0, 18:1 n-9, others) 
concentrations in the chylomicron fraction over 4 hours.  
• Differences in the plasma amino acid (isoleucine, leucine, valine, histidine, lysine, 
methionine, phenylalanine, threonine, alanine, arginine, asparagine, aspartic acid, 
glutamic acid, glutamine, glycine, proline, serine, tyrosine, 3-methyhistidine, citruline, 
hydroxyproline, ornithine, taurine, others) concentrations over the 4 hours.  
• Differences in plasma neurotransmitter concentrations (phenylethyl amine, 3,4-
dihydroxyphenylalanine, dopamine, 3-methoxytyramine, 3,4-dihydroxyphenylacetic 

9 
 
acid, homovanillic acid, norepinephrine, 3,4-dihydroxyphenylglycol, 3-methoxy-4-
hydroxyphenylglycol, normetanephrine, epinephrine, metanephrine, vanillylmandelic 
acid, tryptophan, kynurenine, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic 
acid, alpha-aminobutyric acid, gamma-aminobutyric acid).  
• Differences in minerals and haemoglobin concentrations.  
• Change in inflammatory markers (TNFα, IL-1, IL-6, IL-8, IL-10, IL-13, hs CRP). 
• Digestive differences based on genetic profile. 
• Differences in subjective experience to the meal including fullness, satisfaction, 
gastrointestinal comfort scores among pasture-, grain- and plant-based alternative meat 
meals. 
• Qualitative differences in the participant’s opinions regarding future red meat 
consumption (“Participant insights”). 
• Adverse events, count score differences between meals. 
Measurements 
Blood and Plasma Analyses  
Serum samples will be kept at room temperature for 15 minutes prior to centrifugation and 
storage of supernatant at -80°C until analysis. An aliquot of plasma will be maintained at 4°C 
for the chylomicron-rich fraction (CMRF) separation to occur within 6 h, with the remaining 
stored at -80°C until analysis.  
Fatty acid composition of the CMRF will be analysed by the fatty acid methyl esters (FAME) 
assay as previously described [21] and by lipidomics, as previously described [22].  
Plasma amino acid analysis will be conducted using ultra performance liquid chromatography 
(UPLC) according to previously published methods [23, 24]. The four test meals will also be 
analysed for their final amino acid content. 
The analysis of plasma B-vitamins including thiamine; riboflavin and its vitamer FMN; the B3 
vitamers: nicotinic acid, nicotinamide and nicotinuric acid, and pantothenic acid; and the B6 
vitamers pyridoxal, pyridoxamine, PLP, and 4-pyridoxic acid will be performed by ultra-high-
performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) using a 
method published previously [25], within the Liggins Institute, New Zealand.  
Blood samples will be prepared under the fat-soluble vitamin procedure and analysed using the 
fat-soluble vitamin LC-MS method [26]. Samples will be analysed by AgResearch, New 
Zealand. 
Neurotransmitters will be assessed by mass spectroscopy. The methodology utilises a MS-
probe and stable isotope coding LC MS method developed by AgResearch and has been 
optimised for plasma samples. The protocol aligns with published protocol data [26]. 
Minerals will be assessed using the ICP-MS at Analytica Laboratories, Hamilton, New Zealand 
as per the following protocol, optimised for plasma samples. The samples will be analysed on 
an Agilent 7700 ICP-MS (Agilent Technologies, Wilmington, DE, USA) fitted with an 
ASX500 autosampler (CETAC Technologies, Omaha, NE, USA) and MiraMist nebulizer 
(Burgener Research, Mississauga, ON, Canada). The argon consumption in the torch will be 
20 L/min and the collision cell will be operated with 4 mL/min helium. All consumables will 

10 
 
be supplied by Agilent Technologies. Eighteen elemental species [sodium (Na), magnesium 
(Mg), aluminium (Al), K, calcium (Ca), vanadium (V), chromium (Cr), manganese (Mn), Fe, 
cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), selenium (Se), bromine (Br), silver (Ag), 
antimony (Sb) and barium (Ba)] will be analysed. For the analysis, plasma samples will be 
diluted 25-fold with 2.8% ammonia, 10% isopropanol, 0.2% Triton X solution and 0.1% 
EDTA. Following dilution, an internal standard containing scandium (Sc), germanium (Ge), 
yttrium (Y), rhodium (Rh), terbium (Tb) and iridium (Ir) will be added. A standard curve will 
be generated using five concentrations (0.1, 1, 10, 100 and 1000 μg/L), prepared from Agilent 
Multi-element Calibration Standard 2A (Agilent Technologies, Santa Clara, CA, USA). A 
separate calibrator for bromine (Br) will be prepared from 1000 mg/L KBr (o2si Smart 
Solutions, Charleston, SC, USA). Na, K, Ni and Sb will not be calibrated and these results can 
therefore only be expressed as arbitrary units (AU). Blanks and certified reference serum 
material (Seronorm™ Trace Elements Serum L-1, SERO, Billingstad, Norway) will be used 
throughout the analysis to maintain precision and accuracy. Reference material will be prepared 
according to the manufacturer’s instructions and was diluted in the same manner as the plasma 
samples (i.e., 1 in 25 with ammonia diluent, as described above), and the Seronorm™ values 
will be assigned in accordance with the Essential Requirements (Annex 1 of the IVD Directive 
98/79/EC) and the ISO17511 International standard. Polyatomic interferences were overcome 
because the Agilent 7700 uses the Octopole Reaction System (ORS) with Helium as the 
collision gas. Element isotopes selected were those with the least interference. The Agilent 
Multielement standard 2A is traceable to the NIST Standard Reference Material and was 
produced using ISO9001 quality procedures. 
Plasma inflammatory markers will be analysed in duplicate using the Invitrogen’s 
Inflammation 20-Plex Human ProcartaPlex™ Panel (catalogue number: EPX200-12185-901). 
Briefly, 25 µL of plasma and internal controls will be incubated with magnetic beads prior to 
a series of wash steps. 25 μL of detection antibody will be added and incubated for 30 min 
before 50 μL of Streptavidin-PE was added. The 96-well plate will then analysed using Bio-
Plex® 200 system (BioRad) with inflammatory markers being measured in pg mL−1. 
Plasma glucose and cholesterol (total, LDL, HDL) and triglyceride will be measured using a 
Roche Cobas c311 by enzymatic colorimetric assay and insulin will be measured by an 
electrochemiluminescence immunoassay. 
Saliva analyses 
Saliva samples will be collected in an Oragene ON-500 saliva collection kit and sent to the 
Nutrigenomix laboratory centre at University of Toronto, Canada. The iPLEX Gold assay with 
mass spectrometry-based detection on the Sequenom MassARRAY platform (Agena 
Bioscience, San Diego, CA, USA) will be used for all genotyping. The test will analyse 
variations in 70 genes related to nutrient metabolism, eating habit and food intolerances.  
 
Subjective Analyses  
Satisfaction and gastrointestinal comfort scores will be analysed using a 10 cm visual analogue 
scale, previously validated for use in single meal investigations [27]. Satisfaction scores will 
be completed by the participants upon arrival, once immediately prior to meal ingestion (two 
baseline assessments to account for individual variation), immediately following ingestion, and 
then timed with blood sampling for 4 hours.  

11 
 
Gastrointestinal comfort scores will be analysed by a questionnaire consisting of a series of 
visual analogue scales using intensity anchors (“no symptom”, “the most severe symptom 
imaginable”). Gastrointestinal comfort scores questionnaires will be completed by the 
participants upon arrival, immediately prior to meal ingestion (two baseline assessments to 
account for individual variation), immediately following ingestion of the meal, and then timed 
with blood sampling for 4 hours. 
Participant insights will be assessed via a closed and open-ended questionnaire to ask the 
participant to guess which meat-type they were provided the day prior and the degree to which 
they are likely to consume red meat in the future, with reasoning. The survey will also 
specifically ask for any adverse reactions to the test meal. The participants will be asked to 
complete a survey the day following the final test meal trial, a link to the Qualtrics (SAP, Utah, 
US) survey will be emailed.  
Qualitative Analysis 
Results from the open-ended survey will be transcribed into NVivo (QRS International, 
Victoria, AUS) software and analysed using qualitative techniques for emergent themes. 
DISCUSSION 
Adverse events 
All study-related adverse events will be requested in the post meal survey and will be listed in 
the final trial report. Serious adverse events will be reported to the Health and Disability Ethics 
Committee. 
DECLARATIONS 
Ethics approval and consent to participate 
The investigators will ensure that this study is conducted in accordance with the principles of 
the Declaration of Helsinki, with relevant institutional regulations. There were no funds or time 
allocated for participant involvement in the research design, however we will invite participants 
to assist with our dissemination strategy.  
Consent for publication 
A model consent form is available on the clinical trial registration page. The outcomes will be 
submitted for publication in peer reviewed medical journals and authorship will be determined 
using the guidelines from the International Committee of Medical Journal Editors.  
Trial Steering Committee and Availability of data and materials 
The principal investigator (PI) will be responsible for the project coordination and the research 
support will oversee the operational aspects of the trial. A scientific advisory group will 
regularly monitor the study implementation, as well as the data generation, documentation and 
reporting. Protocol amendments will be communicated by the PI to the ethics committee and 
clinical trial registration. Access to data will be granted to appropriate members of the research 
team and to authorised representatives from the host institution to monitor and/or audit the 
study and ensure compliance with regulations. Data will be made available to external 
academics on reasonable request. 

12 
 
Funding 
This research is funded by the Meat Industry Association Innovation Limited (a subsidiary of 
the New Zealand Meat Industry Association), Beef and Lamb New Zealand Limited and the 
New Zealand Ministry of Business, Innovation and Employment. The funder(s) have no 
involvement in the data collection, analysis and interpretation of the data; in the writing of the 
report; and in the decision to submit the paper for publication. 
Authorship contribution and competing interests 
The employee, employment title, contribution to the research and competing interests have 
been documented for each author in Table 3. 
Table 3: Conflict of Interest Statement 
Author 
Author 
Contributor 
Statement 
Employer 
Financial Conflict 
Intellectual 
Conflict 
Other 
relevant 
disclosures 
A. 
Braakhuis 
Research 
Design, 
Implementation, 
Analysis and 
Reporting 
Research 
Dietitian; 
University of 
Auckland 
As outlined under 
“Funding”. 
No 
Consumes 
3 to 4 
servings of 
both red or 
processed 
meat per 
week 
D. Cameron-
Smith 
Research 
Design 
Collaborating 
Nutritional 
Scientist; 
Agency for 
Science, 
Technology 
and Research,  
DCS has received funding 
from meat companies (e.g. 
Firstlight Foods) to study 
the health impacts on 
consuming red meat 
products 
(ACTRN12618001022257)  
No 
Consumes 
1 to 2 
servings or 
red or 
processed 
meat per 
week 
T. Pham 
Data Collection 
and Analysis 
Research 
Fellow; 
University of 
Auckland 
As outlined under 
“Funding”. 
No 
Consumes 
2 to 3 
servings of 
both red or 
processed 
meat per 
week 
S. Knowles 
Research 
Design, Data 
Analysis and 
Reporting 
Senior 
Research 
Scientist; 
AgResearch 
Ltd 
As outlined under 
“Funding” 
No 
Consumes 
1-2 
servings of 
red or 
processed 
meat a 
week 
M. Barnett 
Research 
Design, Data 
Analysis and 
Reporting 
Senior 
Research 
Scientist; 
AgResearch 
Ltd 
As outlined under 
“Funding”. 
No 
Consumes 
3 to 4 
servings of 
both red or 
processed 
meat per 
week 
L. Kaur 
Research 
Design, Data 
Analysis and 
Reporting 
Senior 
Research 
Officer; 
Massey 
University 
Currently receiving 
funding from MIAI to 
study in vitro digestibility 
of NZ beef in comparison 
No 
Doesn’t 
consume 
meat 

13 
 
with a popular meat 
analogue product. 
E. 
Bermingham 
Research 
Design, 
Implementation, 
Analysis and 
Reporting 
Senior 
Research 
Scientist; 
AgResearch 
Ltd 
ENB is currently receiving 
funding from MIAI to 
investigate the nutrient 
composition of NZ beef. 
Previously she has 
received funding from 
meat companies (e.g. 
Firstlight Foods) to study 
the health impacts on 
consuming red meat 
products 
(ACTRN12618001022257) 
No 
Consumes 
3 to 4 
servings of 
both red or 
processed 
meat per 
week 
 
Trial Status 
Recruitment will commence October 2020.  
 
REFERENCES 
1. Thakur A. Market for Plant-Based Meat Alternatives.  Environmental, Health, and 
Business Opportunities in the New Meat Alternatives Market. IGI Global; 2019: 218-37. 
2. Slade P. If you build it, will they eat it? Consumer preferences for plant-based and cultured 
meat burgers. Appetite 2018;125:428-37.  
3. Circus VE, Robison R. Exploring perceptions of sustainable proteins and meat attachment. 
B Food J. 2019.  
4. Keefe LM. # FakeMeat: How big a deal will animal meat analogs ultimately be? Anim 
Front. 2018;8(3):30-7.  
5. Ekmekcioglu C, Wallner P, Kundi M, Weisz U, Haas W, Hutter H-P. Red meat, diseases, 
and healthy alternatives: A critical review. Crit Rev Food Sci Nutr 2018;58(2):247-61.  
6. Forouhi NG, Krauss RM, Taubes G, Willett W. Dietary fat and cardiometabolic health: 
evidence, controversies, and consensus for guidance. BMJ. 2018;361:k2139.  
7. Hicks TM, Knowles SO, Farouk MM. Global provisioning of red meat for flexitarian diets 
Front Nutr. 2018;5(50). doi:doi: 10.3389/fnut.2018.00050. 
8. Daley CA, Abbott A, Doyle PS, Nader GA, Larson S. A review of fatty acid profiles and 
antioxidant content in grass-fed and grain-fed beef. Nutr J  2010;9(1):10.  
9. McAfee AJ, McSorley EM, Cuskelly GJ, Fearon AM, Moss BW, Beattie JAM et al. Red 
meat from animals offered a grass diet increases plasma and platelet n-3 PUFA in healthy 
consumers. B J Nutr. 2011;105(1):80-9. doi:10.1017/S0007114510003090. 
10. Wang X, Lin X, Ouyang YY, Liu J, Zhao G, Pan A et al. Red and processed meat 
consumption and mortality: dose–response meta-analysis of prospective cohort studies. 
Public Health Nutr. 2016;19(5):893-905.  
11. Zárate R, el Jaber-Vazdekis N, Tejera N, Pérez JA, Rodríguez C. Significance of long 
chain polyunsaturated fatty acids in human health. Clin Transl Med 2017;6(1):25.  
12. Byelashov OA, Sinclair AJ, Kaur G. Dietary sources, current intakes, and nutritional role 
of omega-3 docosapentaenoic acid. Lipid Technol. 2015;27(4):79-82. 
doi:10.1002/lite.201500013. 
13. Bermingham EN, Agnew M, Gomes Reis M, Taukiri K, Jonker A, Subbaraj AK et al. 
Assessment of Atherogenic Index, long-chain omega-3 fatty acid and phospholipid 

14 
 
content of prime beef: a survey of commercially-sourced New Zealand Wagyu and Angus 
beef cattle. Anim Prod Sci 2020;[submitted].  
14. Greco E, Winquist A, Jacob Lee T, Collins S, Lebovic Z, Zerbe-Kessinger T et al. The 
role of source of protein in regulation of food intake, satiety, body weight and body 
composition. J Nutr Health Food Eng. 2017;6(6):00223.  
15. AUS-MEAT Limited. (2010). Australian Beef Carcase Evaluation Beef & Veal Chiller 
Assessment Language. AUS-MEAT National Accreditation Standards and Australian 
Meat Industry Classification System 
https://www.ausmeat.com.au/media/1711/Chiller%2010%20Low.pdf 
16. Chan A-W, Tetzlaff JM, Altman DG, Laupacis A, Gøtzsche PC, Krleža-Jerić K, 
Hróbjartsson A, Mann H, Dickersin K, Berlin J, Doré C, Parulekar W, Summerskill W, 
Groves T, Schulz K, Sox H, Rockhold FW, Rennie D, Moher D. SPIRIT 2013 Statement: 
Defining standard protocol items for clinical trials. Ann Intern Med. 2013;158(3):200-207 
17. Lairon D, Lopez-Miranda J, Williams C. Methodology for studying postprandial lipid 
metabolism. Eur J Clin Nutr 2007;61(10):1145.  
18. Greenheck J, Johnson B, Graves A, Oak A. Giving meat meaning: Creating value-based 
connections with consumers. Anim Front. 2018.  
19. Linderborg KM, Kaur G, Miller E, Meikle PJ, Larsen AE, Weir JM et al. Postprandial 
metabolism of docosapentaenoic acid (DPA, 22: 5n− 3) and eicosapentaenoic acid (EPA, 
20: 5n− 3) in humans. Prostaglandins Leukot Essent Fatty Acids 2013;88(4):313-9.  
20. Dubois CB, G.; Juhel, C.; Armand, M.; Portugal, H.; Pauli, A. M.; Borel, P.; Latgé, C.; 
Lairon, D. Effects of graded amounts (0-50 g) of dietary fat on postprandial lipemia and 
lipoproteins in normolipidemic adults. Am J Clin Nutr 1998;67:31-8.  
21. Milan AM, Nuora A, Pundir S, Pileggi CA, Markworth JF, Linderborg KM et al. Older 
adults have an altered chylomicron response to a high-fat meal. B J Nutr 
2016;115(5):791-9.  
22. Su M, Subbaraj AK, Fraser K, Qi X, Jia H, Chen W et al. Lipidomics of Brain Tissues in 
Rats Fed Human Milk from Chinese Mothers or Commercial Infant Formula. Metabolites 
2019;9(11):253.  
23. Prodhan U, Milan A, Thorstensen E, Barnett M, Stewart R, Benatar J et al. Altered Dairy 
Protein Intake Does Not Alter Circulatory Branched Chain Amino Acids in Healthy 
Adults: A Randomized Controlled Trial. Nutrients 2018;10(10):1510.  
24. Milan A, D’Souza R, Pundir S, Pileggi C, Barnett M, Markworth J et al. Older adults 
have delayed amino acid absorption after a high protein mixed breakfast meal. J Nutr 
Health Aging 2015;19(8):839-45.  
25. Sharma P, Gillies N, Pundir S, Pileggi CA, Markworth JF, Thorstensen EB, Cameron-
Smith D, Milan AM. Comparison of the Acute Postprandial Circulating B-Vitamin and 
Vitamer Responses to Single Breakfast Meals in Young and Older Individuals: 
Preliminary Secondary Outcomes of a Randomized Controlled Trial. Nutrients. 2019 
Dec;11(12):2893. 
26. Khaksari M, Mazzoleni LR, Ruan C, Kennedy RT, Minerick AR. Determination of 
water-soluble and fat-soluble vitamins in tears and blood serum of infants and parents by 
liquid chromatography/mass spectrometry. Experimental eye research. 2017 Feb 
1;155:54-63. 
27. Wong JM, Malec PA, Mabrouk OS, Ro J, Dus M, Kennedy RT. Benzoyl chloride 
derivatization with liquid chromatography–mass spectrometry for targeted metabolomics 
of neurochemicals in biological samples. Journal of Chromatography A. 2016 May 
13;1446:78-90. 

15 
 
28. Flint A, Raben A, Blundell J, Astrup A. Reproducibility, power and validity of visual 
analogue scales in assessment of appetite sensations in single test meal studies. Int J Obes 
2000;24(1):38.