Three sequential studies assessing sugar-free rehydration beverages containing differing amounts of a novel amino acid formula were conducted. All studies received ethical approval from the Loughborough University Ethics Approvals (Human Participants) Sub-Committee (Study 1 ID: LEON3151; Study 2 ID: LEON1416; Study 3 ID: LEON3151-2859) and were registered with Clinical Trials (clinicaltrials.gov; Study 1 ID: NCT04819334; Study 2 ID: NCT04509388; Study 3 ID: NCT05698849). For Study 1 and 2, the amino acid beverages were compared to two commercially available sugar-free beverages, Powerade Zero™(PZ) and Gatorade Zero™ (GZ). For Study 3, the novel amino acid beverage was compared to a commercially available sugar-free rehydration beverage (PZ) and a commercially available 6% carbohydrate–electrolyte beverage (Gatorade Thirst Quencher™; GTQ). In all three studies, subjects completed a screening visit, and three experimental trials commencing at the same time of day (standardised within subjects between 08:00 and 09:00) in a randomised order, separated by ≥ 6 days.

Before commencement of each study, subjects provided written informed consent, consent to publish, and completed a medical screening questionnaire. Subjects were healthy (according to a medical screening questionnaire), non-smokers and had no known history of cardiovascular, metabolic, digestive, or renal disease. During the screening visit, body mass (AFW-120 K, Adam Equipment Co., Milton Keynes, UK) and height (Seca 216, Hamburg, Germany) were measured, whilst body fat was estimated using skinfold measurements (Harpenden Skinfold Caliper, HaB International Ltd., Southam, UK) at the biceps, triceps, sub-scapula and supra-iliac [22], and subjects self-reported their activity levels. All skinfold measurements were taken by the same accredited The International Society for the Advancement of Kinanthropometry (ISAK) anthropometrist. The subject characteristics for the three studies are displayed in Table 1. There was no control for menstrual cycle phase as ovarian hormones/menstrual cycle phase do not appear to affect gastric emptying [23] or hydration outcomes [24].

In each study, subjects completed a diet and physical activity record for the 24 h preceding their first experimental trial and replicated these patterns before the second and third experimental trials. Adherence was verbally checked on arrival for trials. Strenuous exercise or alcohol intake were not permitted during this period. The day before trials, subjects were instructed to consume a minimum of 40 mL/kg body mass of fluid [25, 26]. This volume included any fluid, i.e. water, juice, coffee, tea, carbonated drinks, etc. Subjects stopped eating and drinking at least 10 h before arrival at the laboratory.

Upon arrival at the laboratory, subjects voided their bladder into a plastic container, before nude body mass was recorded, and a flexible 20-gauge cannula was inserted into an antecubital/forearm vein for subsequent blood sampling. Subjects sat on a treatment bed with their legs flat on the bed and the backrest raised at ~ 55° (i.e. a semi-upright Fowler’s position). After 30 min, a baseline blood sample was taken. All blood samples were ~ 7.5 mL, and immediately, following each sample, the cannula was flushed with ~ 7.5 mL isotonic sterile saline (BD Biosciences, New Jersey, USA). A 550 mL (Study 1) or 500 mL bolus (Study 2 and 3) of the experimental beverage was then given, containing 3.000 g of deuterated water (Deuterium Oxide 99.9 atom % D, Sigma-Aldrich, St. Louis, USA), followed by a further 50 mL of the experimental beverage, which was used to swill around the drink vessel to ensure all deuterium oxide was ingested. Subjects were instructed to consume the beverage as quickly as possible, but to prioritise not spilling any. Subjects remained on the treatment bed in the semi-upright Fowler’s position for a further 60 min; the timer began at the commencement of drinking. Additional ~ 7.5 mL blood samples were taken at 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min. A second urine sample was collected after the final blood sample. Ambient temperature and relative humidity (Kestrel 4400, Nielsen-Kellerman Co., Philadelphia, USA) were recorded at 0, 30 and 60 min.

Experimental beverages were administered in a double-blind manner, prepared by an investigator not involved in the data collection or analysis, and served in an opaque bottle. The composition of beverages for the three studies is detailed in Table 2. Protein was calculated from the sum of elemental amino acid gram weights (molecular weight x mM), which included in descending order by concentration, aspartic acid, serine, valine, isoleucine, threonine, and tyrosine. The proprietary amino acid ratios were held constant when adjusting mM and beverage gram weights up or down. The energy density was also estimated from the energy equivalent for whole proteins, allowing for small errors [27].

From each ~ 7.5 mL blood sample, ~ 1 mL was dispensed into a tube containing K2 EDTA (1.75 mg/mL; Teklab, Durham, UK). This was used to determine haemoglobin concentration and haematocrit via the cyanmethemoglobin method and microcentrifugation, respectively. These values were used to estimate changes in plasma volume relative to baseline [28]. These data were collected, and plasma volume estimated, at 10 timepoints in Study 1 (0, 2, 5, 10, 15, 20, 25, 30, 45, 60 min), 14 timepoints in Study 2 (0, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min), and 5 timepoints in Study 3 (0, 5, 15, 30, 60 min). For consistency, plasma volume is displayed at 5 timepoints for each of the three studies (Fig. 3). The reduction of plasma volume data from 10 and 14 timepoints to 5 timepoints in Study 1 and 2, respectively, did not alter the statistical outcomes/findings. From the remaining ~ 6.5 mL of whole blood, ~ 5 mL was dispensed into a second tube containing K2 EDTA (1.6 mg/mL; Sarstedt AG & Co., Nümbrecht, Germany), and ~ 1.3 mL was dispensed into a tube containing lithium heparin (0.25 mg/mL; Sarstedt AG & Co., Nümbrecht, Germany). Plasma was separated from both tubes by centrifugation (2500 g, 20 min, 4 °C) and frozen (− 80 °C) for subsequent analysis. Plasma samples used for D2O enrichment analysis were stored in glass vials.

Freezing-point depression (Gonotec Osmomat 030 Cryoscopic Osmometer; Gonotec, Berlin, Germany) was used to determine the osmolality of plasma from lithium heparin tubes. Urine specific gravity of both baseline and 60 min urine samples was measured on the day of trials (PAL-10S, Digital Urine Specific Gravity Refractometer, Atago Co. Ltd., Tokyo, Japan).

Plasma D2O enrichment was determined in duplicate using the Europa Scientific ANCA-GSL sample preparation unit and 20–20 isotope ratio mass spectrometry (Sercon Ltd., Cheshire, UK). In brief, an appropriate sample volume was pipetted into Exetainer tubes and an insert vial containing 5% platinum on alumina was added. The tubes were sealed and subsequently filled with pure hydrogen. Samples were left for an equilibration period, during which the isotopes in the solution exchanged with the hydrogen gas in the headspace. A sample of the headspace gas was then analysed by continuous-flow isotope ratio mass spectrometry. The isotopic enrichment data are expressed as δ‰ against the international water standard Vienna Standard Mean Ocean Water (VSMOW). The CV of this measurement was 0.23%. Plasma D2O enrichment area under the curve (AUC60) was calculated, and the maximal plasma D2O enrichment concentration observed at any measured time point (Cmax) and the time Cmax occurred (Tmax) were derived [29].

Additional results are provided in the supplementary material for Study 1 and 2 (plasma amino acids [Supplementary Figs. 1 and 2], glucose [Supplementary Fig. 3], lactate [Supplementary Fig. 4], creatinine [Supplementary Fig. 5]), and Study 2 only (plasma sodium [Supplementary Fig. 6A] and potassium [Supplementary Fig. 6B], and urine D2O concentration). Plasma amino acid concentrations were determined at 0, 15, 30, 45 and 60 min using a Biochrom 30 + high-performance liquid chromatography ion exchange system (Biochrom, Cambourne, UK). Plasma glucose, lactate, and creatinine at 0, 15, 30, 45 and 60 min were determined via enzymatic colorimetric method (ABX Pentra C400, Horiba Medical, Northampton, UK). Plasma sodium and potassium were determined at 0, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min via flame photometry (M410C Flame Photometer, Sherwood Ltd., Cambridge, UK). Urine D2O concentration was determined via the method described above for plasma D2O enrichment; pre-trial urine D2O enrichment was subtracted from post-trial urine D2O enrichment to nullify any remaining D2O in the body water pool from previous experimental visits.

Data were initially checked for normality of distribution using a Shapiro–Wilk test. Data containing two factors (Trial*Time) were initially analysed using two-way repeated measures analysis of variance (ANOVA) (SPSS version 27, SPSS Inc., Illinois, USA). Data containing one factor were initially analysed using one-way repeated measures ANOVA (normally distributed data) or Friedman’s ANOVA (non-normally distributed data). Where the assumption of sphericity was violated, the degrees of freedom were corrected using the Greenhouse–Geisser estimate. Significant ANOVA interaction (two-way ANOVA) and main (one-way ANOVA) effects were followed-up by post hoc paired t tests for normally distributed data, and Wilcoxon signed-rank tests for non-normally distributed data. The Holm-Bonferroni correction was applied to post hoc tests to control the family-wise error rate. A a-priori sample size estimation was performed using the data of Hill et al. [30] and Jeukendrup et al. [11], an α of 0.05, and a statistical power of 0.80. It was estimated that 15 subjects would be required per study to reject the null hypothesis for D2O kinetic parameters (e.g. AUC). Statistical significance was accepted when P < 0.05. All data are displayed as mean ± SD.