The Dose-response of Vitamin C-enriched Collagen on Markers of Collagen Synthesis in Middle-Aged Men and Women Following Resistance Exercise
Phase: NA
Conditions: Diet, Food, Nutrition
Sponsor: Rob Erskine

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The Dose-response of Vitamin C-enriched Collagen on Markers of Collagen Synthesis in 
Middle-Aged Men and Women Following Resistance Exercise 
Study protocol, statistical analysis plan and informed consent form 
Document date: 17 January 2024 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Participant Flow 
The number of participants who started and completed this study is shown in Figures 1, and 
2. 
 
 
Figure 1. CONSORT flow diagram for middle-aged males. HC, hydrolysed collagen. 
 
 

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Figure 2. CONSORT flow diagram for middle-aged females. HC, hydrolysed collagen. 
 
 
 
 
 
 
 
 
 
 

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Arm/Group Information 
Arm 1: Intervention for middle-aged male participants (aged 40 – 65 years): Consuming 
hydrolyzed collagen (HC) with resistance exercise. 
Arm 1 Description: 
Middle-aged male participants were asked to visit the laboratory on three different occasions 
to consume one of three different doses of hydrolyzed collagen (HC) and performing 
resistance exercise (RE). Different doses of HC (0 grams, 15 grams and 15 grams) and 50 mg 
vitamin C were dissolved with 250 mL water in an opaque drinks bottle. To match calories of 
30 grams HC, 34.1 grams and 15.4 grams’ maltodextrin were used in the 0 grams HC and 15 
grams HC interventions, respectively. Also, to mask any potential difference in HC doses, 4 
grams’ non-caloric sweetener was included in all HC doses. Each intervention (with a seven-
day washout period) lasted for seven hours and immediately after participants consumed the 
HC, they performed four sets of ten repetitions of leg press exercise at 10-repetition 
maximum load and then rested. Ten x 5 mL blood samples were collected as follows: at rest 
immediately prior to HC ingestion, 0.5 hours post HC ingestion, 1 hour post HC ingestion, 0.5 
hours post exercise, 1 hour post exercise, 2 hours post exercise, 4 hours post exercise and 6 
hours post exercise. All interventions were performed at the same time of day (08:00 - 15:00). 
 
Blood samples were analysed to measure the concentration of a biomarker of collagen 
synthesis (serum procollagen type I N-terminal propeptide (PINP)), a biomarker of collagen 
breakdown (plasma β-isomerized C-terminal telopeptide of type I collagen (β-CTX)), serum 

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collagen amino acid concentration, and concentration of 17β-estradiol (for female 
participants only). 
The details of nutritional supplements used are as follows: 
Hydrolyzed collagen (Myprotein, Cheshire, UK), vitamin C powder (Holland and Barrett 
Retail Limited, Warwickshire, UK), maltodextrin (Myprotein, Cheshire, UK), and non-caloric 
sweetener (Truvia®, SilverSpoon, London, UK). 
Arm 2: Intervention for middle-aged female participants (aged 40 – 65 years): Consuming HC 
with RE. 
Arm 2 Description: 
The intervention procedure is exactly the same as Arm 1 except for the number of visits. 
Middle-aged female participants visited the laboratory on three occasions across two 
consecutive menstrual cycles. The first visit (familiarisation) took place at the onset of 
menses. Following this, there were two interventions within a single month, where female 
participants’ estrogen level was lower (i.e., at the onset of menses) or higher (i.e., at 
ovulation). Dates for the interventions were based on the self-reported onset of menses and 
previous menstrual cycle length. 
Recruitment Period 
Participants were recruited from the local area (Carlow, Ireland). Recruitment began in 
November 2019 and data collection was completed in September 2021. 
 
 

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2. Baseline Characteristics 
Arm 1: Eight middle-aged men (mean ± SD: age, 49 ± 8 years; height, 178 ± 2 cm; body mass, 
90 ± 4 kg). 
Arm 2: Two middle-aged women. One participant (age, 43 years; height, 160.5 cm; body mass 
63.5 kg) was a European masters level karate athlete, while the other was a recreational 
distance runner (age, 42 years; height, 172.6 cm; body mass 69.4 kg). 
• Age 
o Middle-aged participants aged 40 to 65 years 
• Sex 
o Male and female 
• Ethnicity 
o Race and ethnicity information not collected 
• Region of enrolment 
o Carlow, Ireland 
Variables 
Table 1. Primary and secondary outcome measures for each arm of the study. 
Variables 
Study Arm 
Outcome measure type 
Number of 
participants 
analysed 
Serum PINP 
1 and 2 
Primary 
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Plasma β-CTX 
1 and 2 
Primary 
10 
Serum amino acid 
concentration 
1 and 2 
Secondary 
10 
Serum 17β-estradiol 
2 
Secondary 
2 
 

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 3. Outcome Measure Information 
Table 1 shows data for each primary and secondary outcome measure by arm. 
Outcome Measure Title and description 
• Concentration of serum procollagen type I N-terminal propeptide (PINP) (µg/L) 
• Concentration of plasma β-isomerized C-terminal telopeptide of type I collagen (β-
CTX) (µg/L) 
• Concentration of 18 amino acids that constitute collagen (µmol/L): 
Glycine, proline, hydroxyproline, glutamic acid, alanine, arginine, aspartic acid, lysine, 
serine, leucine, valine, phenylalanine, threonine, isoleucine, histidine, tyrosine, 
methionine, and glutamine. 
• Concentration of 17β-estradiol (ng/L) 
Outcome Measure Time Frame 
Outcome measures (table 2) were analysed from August 2022 to November 2022. 
Arm 1 Information 
Arm 1 title: Middle-age male intervention 
Arm 1 description: 
Thirteen men volunteered for this study. Three withdrew prior to the commencement of the 
first intervention. Two participants completed a portion of the study in March 2020, however, 
due to immediate laboratory closure during the COVID-19 lockdown restrictions, they could 
not complete all interventions and were subsequently excluded from the analysis. 
 

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Arm 2 Information 
Arm 2 title: Middle-aged female intervention 
Arm 2 description: 
Fifteen women volunteered for this study. Two female participants completed familiarisation 
and a single intervention each for this study before COVID-19 national lockdown in March 
2020. The remaining volunteers no longer met the inclusion criteria or declined to participate 
once laboratories re-opened in Ireland in July 2021. 
In Arm 1, data from 8 male participants were analysed and in Arm 2, data from 2 female 
participants were analysed.  
Outcome Measure Data Table 
Table 2. Outcome measure type and measure of dispersion by arm. 
 
 
 
 
 
Arm 1 
Variable 
Measure 
type 
Measure of dispersion 
Serum PINP (µg/L) 
Mean 
Standard deviation 
Plasmaβ-CTX (µg/L) 
Mean 
Standard deviation 
18 Serum amino acids (µmol/L) 
Mean 
Standard deviation 
Arm 2 
Serum PINP (ng/mL) 
n/a 
n/a 
Plasmaβ-CTX (µg/L) 
n/a 
n/a 
18 Serum amino acids (µmol/L) 
n/a 
n/a 
Serum 17β-estradiol (ng/L) 
n/a 
n/a 

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Statistical Analyses 
Due to the sample size (n=2) in Arm 2, only data from Arm 1 were analysed with statistical 
models. The data from Arm 2 were analysed descriptively.  
Two-way within-subject ANOVA models (dose × time) were performed to detect changes in 
serum PINP, amino acid and plasma β-CTX concentration. One-way repeated measures 
ANOVA models were performed to detect differences in the area-under-the-curve between 
interventions for PINP and each of the 18 amino acids. If an interaction effect or a main effect 
of HC dose existed, least significant difference post-hoc pairwise comparisons were 
performed. The level of statistical significance was set at P < 0.05. 
4. Adverse Event Information 
During the data collection period, any adverse event such as all-cause mortality or serious 
adverse event did not occur. 
5. Certain Agreements 
Are all PIs Employees of Sponsor? 
Yes: The principal investigator is an employee of the sponsor 
Results Disclosure Restriction on PI(s)? 
No 
6. Results Point of Contact 
Dr Rob Erskine 
Research Institute for Sport and Exercise Sciences, Liverpool John Moores University 
Tel: +44 (0)151 904 62; E-mail: R.M.Erskine@ljmu.ac.uk 

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Informed Consent Form