This study aims to address two key aspects. First, the suitability of selecting a specific sampling site for BHB measurement in patients and research, as well as potential differences between capillary and venous blood measurements. Additionally, the study will investigate the effects of ketosis on EPO concentrations, sex hormone levels, hemodynamic markers, and blood pressure.

This investigation will utilize blood samples collected from baseline and at Day 15, between which participants are exposed to intermittent ketosis or placebo in a randomized parallel design.

Aim and Perspective The primary objective of this study is to ascertain The agreement between estimates of beta-hydroxybutyrate (BHB) in capillary and venous blood and whether this agreement is influenced by the level of BHB.

The agreement in BHB measurements between finger and earlobe capillary samples. The agreement in BHB measurements between venous estimates obtained through a point-of-care device (KetoSure) and full blood estimates obtained through hydrophilic interaction liquid chromatography tandem mass spectrometry (HLCMS).

A central aim of the study is also to investigate The impact of ketosis on short-term and longer-term erythropoietin (EPO) levels.

The resulting effects of EPO on erythropoiesis and iron metabolism during a two-week period of intermittent ketosis.

The effects of ketosis on sex hormones including derived factors. The effects of ketosis on hemodynamic markers and blood pressure.

The study aims to determine the appropriateness of selecting a specific sampling site for BHB measurement in both patient care and research. Additionally, it seeks to identify any differences between BHB measurements from capillary and venous blood samples. The study will also examine the concordance between the KetoSure point-of-care-test (POCT) device and the established gold standard, offering insight into any discrepancies arising from electrochemical estimations and HLCMS.

Accurate BHB measurement is crucial in clinical and experimental settings. Firstly, precise BHB quantification can inform clinical decision-making for conditions such as suspected hyperinsulinemia, uncertain etiology hypoglycemia, and diabetic ketoacidosis. Secondly, given the extensive research on BHB inference and ketones in recent years, the credibility of these studies heavily hinges on the precision of measurements concerning sample type and sampling site selection.

During the randomized study period, the effects of ketosis on EPO concentrations, sex hormone levels, hemodynamic markers, and blood pressure measurements will be explored. These analyses will be conducted using blood samples collected from baseline through the acute study visit as well as on Day 15, when participants receive intermittent BHB ketone ester supplementation or matched placebo according to randomization.

Analytical approach Following a visual assessment of graphical linearity representation, differences will be calculated using the paired t-test, agreement determined using the Bland-Altman plot, and correlations assessed using Pearson's r. Further calculations will employ Lin's concordance correlation coefficient of absolute agreement. An analysis equivalent to repeated measurements ANOVA will be applied. No imputation of missing data will be conducted, and steps will be taken to ensure data completeness before participants leave the research facilities.

Sample Size and Power Calculation Given the absence of prior studies on BHB agreement data, our study's sample size calculations are based on relevant literature. Citing Boyd et al., and considering correlated glucose estimates, a sample size of 13 participants will provide sufficient statistical power (alpha = 0.05, beta = 20%) to detect a difference of 0.58 mM in glucose estimates between capillary and venous blood samples (SD = 0.68 mM). A sample size of 20 participants is justifiably required to detect a clinically significant difference of 1 mM (SD = 1.5) under the same parameters. Consequently, we will include 16 patients in our study.

Up to 150 mL of blood will be collected during the study, including repeated sampling through an indwelling catheter during acute testing and follow-up outpatient sampling at Day 15.

Purpose of Storage Biological samples will be stored at -80 °C for the duration of data collection and for 18 months thereafter to allow for batch analyses. A research biobank will be established for samples not analyzed immediately, with surplus material preserved for potential future research.

Handling of Patient Information Access to electronic health records is limited to laboratory results necessary for study analyses and is included in informed consent. Data will be pseudonymized during analysis. De-identification codes will be retained by the primary investigator.

Data Privacy and Sharing Data access is restricted to investigators until completion of final analyses. After anonymization, data may be shared upon reasonable request. Public data sharing is under consideration following publication.

Financial Information The study is investigator-initiated and funded through independent research funds within the Department of Internal Medicine, Regional Hospital Viborg. No investigators have financial interests in the intervention.

Inclusion Criteria:

• Age 18-60 years
• BMI 19-30 kg/m2
• Expected ease of catheter insertion
• Considered of sound health
• Oral and written informed consent

Exclusion Criteria:

• Inability to fully understand the consent including consent forms
• Inability to cooperate to the study
• electrolyte disorders
• acute or chronic kidney disease or ompromised renal function including excess risk
• servere hypertension
• autoimmune disease
• liver or bile disease
• diabetes mellitus
• reactive hypoglycemia or similar disorders
• treatment with drugs, and dietary supplements with inference on key metabolic or hormonal markers, e.g. insulin, glucagon, DDP-IV inhibitors, GLP-1 RA, sulfunylurea
• use of illegal or otherwise use of medicinal products without prescription
• anemia or other know disease of the hematopoietic system
• previous bariatric surgery
• previous myocardial infarction or uncontrolled myocardial ischemia
• recent intended/unintended weight loss
• allergies to catheters or adhesives

Acute study visit and Baseline to Day 15

Adverse events were collected during a supervised acute BHB ketone ester exposure prior to randomization and during the subsequent randomized parallel treatment phase (Baseline to Day 15). Events are reported according to exposure period.

The study was adequately powered for erythropoietin outcomes but had limited power for sex-specific analyses and downstream erythropoietic endpoints due to sample size and duration.

Participants are blinded to the drink (intervention) being either ketosis (obtained by ingestion of a ketone monoester drink) or placebo (a taste and volume matched drink). Drinks are provided in similar neutral unlabelled bottles