Post Exercise Hydration Responses to an Electrolyte, Glycerol, and Creatine Supplement in Horses: A Preliminary Study

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However, limited data are available on formulations that include osmotically active compounds such as glycerol and creatine. These additional ingredients may enhance fluid retention, yet their physiologic effects in equine athletes have not been evaluated. This preliminary study assessed hematologic responses, body mass recovery, and voluntary water intake following moderate exercise with and without supplementation using a commercially available hydration supplement containing electrolytes, creatine, and glycerol. Six healthy quarter horses completed two conditions in a cross-over design: a water-only condition and a supplemented condition. Each study day consisted of a uniform moderate exercise regimen, a recovery period, and a 3-hour hydration period. Hematocrit and hemoglobin decreased over time in both conditions, while total protein concentrations were consistently lower in the supplemented condition. Calculated plasma volume (PV) increased to a greater extent with supplementation (p=0.033). Body mass was preserved during the hydration period in the supplemented condition but declined in the water condition (p = 0.004). Horses also voluntarily consumed more water when supplemented (p = 0.0079). These preliminary findings suggest that an electrolyte supplement containing glycerol and creatine may augment post-exercise rehydration by promoting increased water intake, supporting PV expansion, and improving body mass recovery in moderately exercised horses. Horse electrolytes hydration creatine glycerol Figures Figure 1 Figure 2 Figure 3 Introduction Hydration is a primary determinant of thermoregulatory capacity and exercise tolerance. During prolonged or moderately intense work, especially in hot environments, horses can lose 10–15 L of fluid per hour through sweat, rapidly reducing plasma volume (PV) and compromising thermoregulatory capacity ( 1 ). This sweat loss includes concentrations of sodium, chloride, potassium, and other electrolytes, resulting in a combined water and electrolyte deficit that can compromise cardiovascular stability, neuromuscular function, and recovery ( 1 – 7 ). Furthermore, working horses experience markedly elevated sodium and chloride changes compared with maintenance, and these electrolyte losses increase in proportion to workload and ambient temperature ( 8 ). Effective post-exercise fluid recovery depends on replenishing both water and electrolytes, because electrolyte replacement supports osmotic regulation and improves net fluid retention. ( 7 , 9 ). Oral electrolyte solutions are widely utilized in equine sports medicine because they enhance intestinal ion uptake, promote osmotic movement of water into the extracellular fluid compartment, and support restoration of PV ( 7 , 9 – 11 ). Electrolyte mixtures can also stimulate voluntary drinking behavior, improving total fluid intake during recovery and contributing to more complete rehydration ( 11 , 12 ). Collectively, these data highlight the importance of electrolyte-based hydration strategies in horses undergoing moderate to intense work resulting in fluid loss through evaporative cooling. Beyond traditional electrolyte formulations, glycerol has been evaluated as an osmotic agent capable of inducing “hyperhydration” states in humans and equines. In humans, ingesting glycerol with water increases plasma osmolality, reduces urine output, and expands total body water for several hours, thereby enhancing fluid availability during subsequent exercise ( 13 , 14 ). Similar observations have been reported in horses, where nasogastric administration of glycerol solutions produces transient hyperhydration characterized by increased renal water conservation and greater voluntary water intake ( 15 ). Together these findings suggest that glycerol can augment oral rehydration strategies by increasing the proportion of ingested water that is retained rather than excreted, potentially contributing to improved cardiovascular and thermoregulatory stability during recovery. Creatine supplementation represents another strategy to influence fluid distribution and retention as an ergogenic aid. In humans, creatine loading protocols consistently increase muscle creatine content and are associated with increases in total body water and intracellular water ( 16 ). A substantial body of literature supports the safety and efficacy of creatine supplementation across athletic and clinical populations, with no evidence of harmful fluid shifts when taken at standard doses ( 17 ). More recent reviews emphasize creatine’s potential role in supporting cellular hydration, though no research has evaluated creatine’s hydration-related effects in horses, representing a meaningful gap in equine physiology. Together, these findings suggest that horses can experience large fluid and electrolyte losses via sweat, which can challenge re-hydration and recovery. Furthermore, oral electrolyte solutions enhance fluid intake and promote plasma volume restoration, whereas glycerol and creatine may further support fluid retention via osmotic mechanisms and increases in total body and intracellular water content, respectively. However, this combination of ingredients has not been tested as a hydration supplement in equine athletes. The purpose of this preliminary investigation was to evaluate the effects of a novel oral electrolyte supplement containing glycerol and creatine on post-exercise fluid consumption, body mass recovery, and hematologic markers of hydration in horses. We hypothesized that, compared with water alone, the electrolyte, glycerol, and creatine supplement would: ( 1 ) increase voluntary water intake during the post-exercise hydration period, ( 2 ) enhance water retention as assessed by recovery of body mass, and ( 3 ) promote greater intravascular fluid expansion. Materials and Methods Animals Six Quarter Horses (three mares and three castrated males) from a privately managed training facility were enrolled. All horses were deemed healthy based on physical examination by a licensed veterinarian (DVM). Mean baseline body mass was 506 kg (range 463–566 kg). Horses were maintained under consistent husbandry practices, including a routine morning grain meal prior to each day’s data collection. Horses also received a half flake of alfalfa hay during the 3-hour hydration period on both study days. As all procedures consisted of routine exercise, standard handling, and non-invasive jugular venipuncture performed by a veterinarian as part of normal clinical monitoring, Institutional Animal Care and Use Committee (IACUC) approval was not required. This was consistent with American Veterinary Medical Association guidelines for observational studies conducted within the scope of ordinary veterinary care. Owner consent was obtained for participation. Study Design A crossover, repeated-measures design was used, with each horse completing two experimental conditions on consecutive days. On Day 1 (water condition), horses were exercised and then provided free-choice access to plain water during a 3-hour hydration period. On Day 2 (electrolyte condition), horses followed the same protocol except they received an oral electrolyte paste supplement (100X Equine, HydraMax™) containing electrolytes, creatine, and glycerol, administered at the start of the hydration window. Blood sampling, body mass measurements, and water intake assessments were performed in the same order and at equivalent timepoints relative to exercise on both days. All data were collected in the morning following normal feeding to minimize diurnal variation. Data collection occurred between 10:00 a.m. and 3:30 p.m. Ambient temperature ranged from 20.6–31.1°C with mean relative humidity of approximately 67%, and conditions were consistent across study days. Exercise Protocol Each horse completed a standardized 20-minute lunge line exercise session designed to elicit a moderate cardiovascular and musculoskeletal workload, consistent with typical training. The protocol consisted of 10 minutes of trotting (5 minutes each direction), followed by 10 minutes of loping (5 minutes each direction). Horses were not tacked and were exercised in a halter attached to a lunge line in a covered outdoor arena with sand–fiber footing. Following exercise, horses completed a 30-minute cool-down period consisting of hand walking and standing rest prior to post-exercise measurements. Treatment Conditions During the water condition, horses had ad libitum access to plain water for 3 hours, provided in two buckets. Each bucket contained 14.4 kg of water (28.8 kg total available). No supplement was administered. During the electrolyte condition, horses received one full serving of the electrolyte paste supplement according to manufacturer instructions immediately prior to ad libitum access to plain water for 3 hours, provided in the same manner as the water condition. Blood Sampling and Analysis Blood samples were collected at five timepoints relative to exercise: baseline (pre-exercise), immediately following the cool-down period (Post-Exercise), and at 1, 2, and 3 hours of hydration (1-Hour, 2-Hour, and 3-Hour). Samples were obtained via jugular venipuncture using sterile technique and collected into EDTA tubes, then refrigerated until analysis. All samples were processed within 24 hours at an Antech Diagnostics Veterinary Laboratory. Measured variables included hematocrit, hemoglobin, total plasma protein, and fibrinogen. Percent change in plasma volume (PV) was calculated using the Dill and Costill Eq. (18). Body Mass and Water Intake Body mass was measured at baseline, post-exercise, and at the end of the 3-hour hydration period using a platform livestock scale (Model 244244, Global Industrial, Port Washington, NY, USA; capacity 1000 kg; readability 0.45 kg). The scale was calibrated by the user according to manufacturer instructions prior to data collection. Horses were weighed wearing a halter only, and consistent positioning was maintained across measurements. Water buckets were weighed immediately before and after the hydration period using a portable medical scale (Model SF-891, VEVOR, Kent, WA, USA; capacity 180 kg; readability ± 0.05 kg). Water intake was calculated as the difference between pre- and post-hydration bucket weights, assuming a density of 1.0 kg/L. Statistical Design A two-way repeated-measures analysis of variance (ANOVA) was used to evaluate the effects of Condition (Water, Electrolyte), Time, and their interaction on hematologic variables (hematocrit, hemoglobin, total protein, and fibrinogen) and on body mass. For hematologic outcomes, three timepoints were analyzed: baseline, post-exercise, and the mean of the 1-, 2-, and 3-hour post-hydration samples. Percent change in plasma volume (ΔPV) could only be calculated for the post-exercise and post-hydration timepoints; therefore, a 2 × 2 repeated-measures ANOVA was used for ΔPV, with Condition and Time as within-subject factors. When a significant Condition x Time interaction was detected, Bonferroni-adjusted paired post hoc comparisons were performed to identify differences between conditions and/or timepoints. Water intake was measured once per condition and compared using paired-samples t-tests. All analyses were performed within subject, with horse treated as the repeated factor. Statistical analyses were conducted using GraphPad Prism (Version 10). Results Hematologic Measures All hematological data are presented in Table 1 . Baseline hematocrit did not differ between the water and electrolyte conditions (p = 0.31). Hematocrit changed significantly over time (p = 0.0014), with no main effect of condition (p = 0.72) and no Condition x Time interaction (p = 0.18). The baseline to post-exercise change did not differ between conditions (p = 0.11). Baseline hemoglobin did not differ between conditions (p = 0.30). Hemoglobin demonstrated a significant time effect (p = 0.003), with no main effect of condition (p = 0.47) and no Condition x Time interaction (p = 0.41). The baseline-to-post-exercise change did not differ between conditions (p = 0.27). Baseline plasma protein did not differ between conditions (p = 0.19). Plasma protein post-exercise differed between conditions (p = 0.00016) and changed over time (p = 0.0061), with no Condition x Time interaction (p = 0.26). The baseline-to-post-exercise change did not differ between conditions (p = 0.16). Baseline fibrinogen did not differ between conditions (p = 0.27). Fibrinogen did not differ by condition (p = 0.70), time (p = 0.94), or Condition x Time interaction (p = 0.28). The baseline-to-post-exercise change did not differ between conditions (p = 0.26). Calculated percent change in plasma volume (ΔPV) differed between conditions (p = 0.033) and changed over time (p = 0.0078), with no Condition x Time interaction (p = 0.99) (Fig. 1 ). Mean ΔPV during the post-hydration period did not differ significantly between conditions (p = 0.095). Table 1 Hematologic values across time and stratified by condition. Phase Condition Hct (%) Hgb (g/dL) Plasma Protein (g/dL) Fibrinogen (mg/dL) ΔPV (%) Baseline Water 40 ± 1.8 (35.0–44.3) 13.0 ± 0.7 (11.2–14.9) 6.6 ± 0.1 (6.2–7.0) 158.0 ± 11.9 (127.4–188.6) N/A Electrolytes 41 ± 2.2 (35.1–46.2) 13.4 ± 0.8 (11.3–15.5) 6.5 ± 0.2 (6.1–6.9) 152.5 ± 12.1 (121.3–183.7) N/A Post-Exercise Water 41 ± 1.3 (37.4–44.0) 13.4 ± 0.4 (12.2–14.5) 6.7 ± 0.2 (6.2–7.2) 153.5 ± 10.5 (126.5–180.5) –3.8 ± 4.3 (–14.9–7.3) Electrolytes 41 ± 1.5 (36.8–44.5) 13.4 ± 0.6 (11.8–15.0) 6.4 ± 0.1 (6.0–6.8) 159.3 ± 15.4 (119.7–199.0) 0.0 ± 3.8 (–9.8–9.8) Hydration Water 37 ± 0.9 (34.6–38.6) 12.0 ± 0.3 (11.3–12.7) 6.4 ± 0.1 (6.2–6.6) 159.7 ± 7.3 (144.3–175.1) 14.7 ± 2.0 (10.5–18.8) Electrolytes 37 ± 1.1 (34.3–38.7) 12.1 ± 0.4 (11.4–12.9) 6.2 ± 0.1 (6.0–6.4) 151.6 ± 7.5 (135.8–167.4) 18.5 ± 2.9 (12.4–24.5) Statistical Effects † † †‡ — †‡ Hematocrit (Hct), hemoglobin (Hgb), plasma protein, fibrinogen and percent change in plasma volume (ΔPV) across Baseline, Post-Exercise, and Hydration periods for Water and Electrolyte conditions. Values presented as Mean ± SE (95% confidence intervals). Hydration values represent the average of the 1-, 2-, and 3-hour post-exercise hydration timepoints. † Significant main effect of Time (p < 0.05); ‡ Significant main effect of Condition (p < 0.05). **Table 1 . Approximately here** **Figure 1 . Approximately here** Body Mass and Water Intake Baseline body mass did not differ between the water and electrolyte conditions (p = 0.74), and post-exercise mass also did not differ between conditions (p = 0.54). A significant Condition x Time interaction was detected for body mass (p = 0.004), and a time effect was observed (p = 0.043). At the end of the hydration period, body mass differed between conditions (p = 0.0219) demonstrating that the supplemented condition resulted in a greater preservation of body mass than water alone. Voluntary water intake during the hydration period was significantly greater in supplemented horses than in the water condition (p = 0.0079). **Figure 2 . Approximately here** **Figure 3 . Approximately here** Discussion Data from this preliminary study suggest that oral administration of an electrolyte supplement containing glycerol and creatine prior to ad libitum water access may improve post-exercise hydration compared with water alone. Horses receiving the supplement consumed more water, recovered a greater proportion of post-exercise body mass, and exhibited greater calculated PV values at each timepoint. Electrolyte supplementation is a well-established strategy to replace sweat losses and support hydration in equine athletes. Horses can lose large volumes of hypertonic sweat during exercise, resulting in substantial sodium, chloride, and potassium depletion, altered plasma osmolality, and reduced thirst drive if electrolytes are not replaced adequately ( 3 , 5 , 6 , 8 , 19 ). In the present study, the supplemented condition showed greater increases in PV, which is consistent with improved intravascular fluid delivery. Similar improvements in circulating blood volume, voluntary water intake, and recovery from dehydration have been reported in equines receiving electrolyte-containing formulations following endurance exercise bouts ( 7 , 11 , 12 ). The current data suggest that, even following moderate lunge-line exercise, electrolyte replacement combined with glycerol and creatine may enhance post-exercise fluid recovery and help reduce cardiovascular and thermoregulatory strain in subsequent work. The addition of glycerol may have acted synergistically with electrolytes to enhance fluid retention and therefore restoration of body mass post-exercise. Glycerol is an osmotic compound that has the capability to increase total body water and reduces renal free-water clearance, thereby prolonging the retention of ingested fluids ( 13 – 15 , 20 ). In this preliminary study, horses in the supplemented condition recovered more of their lost body mass than in the water condition, and in several cases approached or exceeded baseline mass by the end of the hydration period. This suggests a net positive water balance with supplementation of the novel electrolyte supplement. Previous research in horses has demonstrated that glycerol containing solutions can induce transient hyperhydration, increase water intake, and enhance renal water conservation ( 15 ). Similarly, human research has shown that glycerol solution ingestion can increase total body water, sustain hyperhydration, and may reduce thermoregulatory and cardiovascular strain during prolonged exercise or heat exposure ( 13 , 14 , 20 – 22 ). Recent research in runners further confirms that glycerol hyperhydration improves fluid retention and can positively influence thermal responses ( 23 ). Although urine output was not measured in the present study, the observed pattern of body-mass recovery is consistent with these osmotic and renal effects. Creatine supplementation may also have contributed to the improved hydration profile, likely through effects on intracellular water. Creatine is taken up into skeletal muscle via a sodium-dependent transporter, increasing intracellular osmolality and drawing water into the cell ( 16 , 17 , 24 – 26 ). In humans, creatine loading reliably increases total body water and lean mass, with a substantial component of this increase attributed to intracellular water expansion ( 16 , 24 , 26 ). Reviews and organizational position statements support the safety of creatine supplementation and highlight its consistent effects on cellular hydration and muscle energetics ( 17 , 25 , 26 ). Although equine specific data on creatine use are limited, the combination of creatine with electrolytes and glycerol in the present formulation may have provided an additional stimulus for water retention. Given that both body mass and calculated PV improved beyond water alone, the overall hydration response is consistent with a supplement acting across multiple fluid compartments, even if the precise intracellular contribution in horses remains to be determined. Substantial individual variability was evident in the hydration response to electrolyte supplementation. One horse in particular displayed a relatively larger improvement during the electrolyte condition than the group mean, indicating an augmented physiological sensitivity to osmotic and electrolyte driven hydration mechanisms. During the hydration period, water intake was nearly twofold greater with electrolyte supplementation (14.8 kg consumed) compared to water alone (7.6 kg consumed). This intake was accompanied by an approximately threefold greater recovery of body mass relative to the water condition (+ 6.5 kg electrolyte vs. − 3.5 kg water). Plasma volume expansion was also 1.5-fold greater with supplementation, suggesting enhanced intravascular rehydration compared to water alone. These augmented responses in a single horse suggest that some horses possess an inherently stronger drive to drink or more pronounced renal and cardiovascular sensitivity to osmotic changes, leading to a more robust fluid retention profile when electrolytes and osmolytes are provided. Such inter-individual variation has been reported in equine hydration research and likely reflects differences in sweat losses, thirst perception, renal concentrating capacity, and overall fluid-regulatory physiology ( 6 , 10 , 12 , 19 ). The present findings emphasize that certain horses may derive markedly greater benefit from electrolyte containing supplements, reinforcing the need to consider individual responsiveness when developing hydration strategies. The present results are broadly consistent with prior work demonstrating that formulations containing electrolytes alone or in combination with glycerol can enhance water intake, support PV recovery and reduce indices of dehydration in horses ( 7 , 11 , 12 , 14 , 15 ). What distinguishes this preliminary study is the inclusion of creatine and glycerol within an equine targeted supplement and the focus upon moderate intensity, routine exercise rather than only high-intensity or long duration endurance workloads. Many performance horses perform repeated, submaximal sessions rather than prolonged competition efforts. The initial finding that water alone did not fully restore hydration status, whereas the supplement did, is practically relevant for daily training management. By improving both fluid intake and retention, such supplementation may help maintain more stable hydration across training days and reduce the cumulative strain of repeated mild-to-moderate dehydration. Additionally, no adverse effects of this electrolyte, glycerol, and creatine supplement were observed during the study, nor upon the subsequent days. Several limitations should be acknowledged. First, fluid compartments were not directly measured. Thus, intracellular versus extracellular water distribution cannot be distinguished, and conclusions about compartment specific shifts remain inferential. Second, urine volume and urinary electrolyte excretion were not assessed, which limits interpretation of renal responses to glycerol and electrolytes. Third, the sample size (n = 6) is modest for a full research study, and may limit the detection of smaller effects. Finally, the fixed order of conditions (water day followed by supplement day) introduces potential sequence effects, although the within-subject design reduces inter-individual variability and strengthens internal validity. In summary, an electrolyte, glycerol, and creatine supplement administered prior to free water access enhanced post-exercise rehydration compared with water alone in moderately exercised horses. The supplement increased voluntary drinking, improved body-mass recovery, and produced greater calculated PV expansion, indicating more effective hydration. These preliminary findings support the use of combined electrolyte, creatine, and glycerol formula to improve post-exercise fluid recovery in equine athletes and justify further research to validate these initial findings. Further research studies are planned to address the limitations of the current preliminary study and to assess the relative efficacy of electrolytes alone compared to electrolytes with the addition of creatine and glycerol. Furthermore, research is planned to inform the development of guidelines for hydration management of horses exercising or training consistently in hot and humid environments. Declarations Author Contributions Author Contributions: Conceptualization, R.P. and J.D.; methodology, R.P.; formal analysis, R.P.; investigation, R.P.; writing: original draft preparation, R.P.; writing: review and editing, J.D.; supervision, J.D. All authors have read and agreed to the published version of the manuscript. Contributing author: Dr. Jacquelyn Dietrich [email protected] Institutional Review Board Statement Ethical review and approval were not required for this study given that all procedures were performed by a licensed veterinarian during standard training and monitoring. Informed Consent Statement Owner consent was obtained prior to horse enrollment. Competing Interests The authors declare that they have no competing interests. Funding This research received no external funding. The electrolyte supplement used in this study was provided by 100X Equine. All remaining study costs were covered by the participating horse owners. Data Availability Statement The data supporting the findings of this study are available from the corresponding author upon reasonable request due to patient/owner privacy. Acknowledgments The authors gratefully acknowledge Abigail Nesbit and Megan Thelen for their significant contributions to this project. Their commitment to horse safety and their attention to detail greatly enhanced the quality of data collected. Conflicts of Interest The authors declare no conflict of interest. 100X Equine provided the electrolyte supplement used in this study but had no role in study design, data collection, data analysis, manuscript preparation, or the decision to publish. References Flaminio MJ, Rush BR. Fluid and electrolyte balance in endurance horses. Vet Clin North Am Equine Pract. 1998 Apr;14(1):147–58. Lindinger MI. Oral Electrolyte and Water Supplementation in Horses. Vet Sci. 2022 Nov 10;9(11):626. McCutcheon LJ, Geor RJ, Hare MJ, Ecker GL, Lindinger MI. Sweating rate and sweat composition during exercise and recovery in ambient heat and humidity. Equine Vet J Suppl. 1995 Nov;(20):153–7. Kerr MG, Snow DH. Composition of sweat of the horse during prolonged epinephrine (adrenaline) infusion, heat exposure, and exercise. Am J Vet Res. 1983 Aug;44(8):1571–7. Kingston JK, Geor RJ, McCutcheon LJ. 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International Society of Sports Nutrition position stand: safety and efficacy of creatine supplementation in exercise, sport, and medicine. J Int Soc Sports Nutr. 2017;14:18. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 20 Apr, 2026 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 21 Feb, 2026 Editor assigned by journal 16 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 14 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8882565","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594905945,"identity":"38a352d9-3b34-4854-8378-18d5a044587a","order_by":0,"name":"Russell Peterson","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYJCCAxUGQJK9sfFBQgUDgwFRWs6AlPEcPmzw4QyRWhjOgAiJtDTJmW1EaDFnP/zwwIECOzm+AzkG0rzzDsubszcfYPhRsQ2nFsueNIMDBwySjSWBzjPm3XbYcGfPsQTGnjO3cWoxOJDDcPiDwYHEDQd7DJKBWhg33MgxYGZsw6Pl/BsGoC0H6jcc5jE4zDvnsD1hLTdywFoSDI6xJTbObDicSFCL5YxnYL8YzjzDfJjhw7H05A1njiUcxOcXc/7kxx8O/LGT57v/sP1HQo217YbjzQcf/KjA4zA46wCYbEZiE6mlDp/iUTAKRsEoGKEAAAhYaVO+4e2zAAAAAElFTkSuQmCC","orcid":"","institution":"Independent Researcher","correspondingAuthor":true,"prefix":"","firstName":"Russell","middleName":"","lastName":"Peterson","suffix":""},{"id":594905946,"identity":"ac2687ea-571c-4b88-92bd-37fa7898d621","order_by":1,"name":"Jacquelyn Dietrich","email":"","orcid":"","institution":"Independent Researcher","correspondingAuthor":false,"prefix":"","firstName":"Jacquelyn","middleName":"","lastName":"Dietrich","suffix":""}],"badges":[],"createdAt":"2026-02-14 21:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8882565/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8882565/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-026-05493-w","type":"published","date":"2026-04-20T15:59:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":103309212,"identity":"7328f196-fbf4-4047-a76c-a14eb50c886b","added_by":"auto","created_at":"2026-02-24 09:49:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":104287,"visible":true,"origin":"","legend":"\u003cp\u003ePercent change in plasma volume (ΔPV) from baseline at Post-Exercise and Hydration time points in the Water (green) and Electrolyte (orange) conditions. Thin lines connect paired data from individual horses (n = 6); thick lines with error bars denote group mean ± SE. Horizontal bracket indicates within-condition comparisons across time points; the vertical bracket indicates the between-condition comparison. No condition x time interaction was observed. *p\u0026lt;0.05; **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8882565/v1/0c82424163f9eb78f94de05a.png"},{"id":103309213,"identity":"740d867a-da09-4531-9ba1-54d64aa264a0","added_by":"auto","created_at":"2026-02-24 09:49:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":70037,"visible":true,"origin":"","legend":"\u003cp\u003eBody mass at Baseline, Post-Exercise, and Hydration time points in the Water (green) and Electrolyte (orange) conditions. Thin lines connect paired data from individual horses (n = 6); thick lines with error bars denote group mean ± SE. A significant Condition x Time interaction was observed; post-hoc between-condition comparisons are indicated at each time point. *p\u0026lt;0.05; ns = not significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8882565/v1/2fbcd56638df822b02ea4ee8.png"},{"id":103309214,"identity":"d47ff459-0b57-4e15-8e5d-ca186ac59558","added_by":"auto","created_at":"2026-02-24 09:49:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49994,"visible":true,"origin":"","legend":"\u003cp\u003eWater intake during the 3-hour hydration period between Water (green) and Electrolyte (orange) conditions. Thin gray lines connect paired data from individual horses (n = 6); thick lines with error bars denote group mean ± SE. Horizontal bracket indicates the between-condition comparison. **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8882565/v1/752aee2296f62798237ec1c2.png"},{"id":107928109,"identity":"85ead551-4fe7-4665-a4ec-71f69c1ea635","added_by":"auto","created_at":"2026-04-27 16:08:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":407159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8882565/v1/434b27b9-8e71-4bc7-a2f1-5e0d5ad99753.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Post Exercise Hydration Responses to an Electrolyte, Glycerol, and Creatine Supplement in Horses: A Preliminary Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydration is a primary determinant of thermoregulatory capacity and exercise tolerance. During prolonged or moderately intense work, especially in hot environments, horses can lose 10\u0026ndash;15 L of fluid per hour through sweat, rapidly reducing plasma volume (PV) and compromising thermoregulatory capacity (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). This sweat loss includes concentrations of sodium, chloride, potassium, and other electrolytes, resulting in a combined water and electrolyte deficit that can compromise cardiovascular stability, neuromuscular function, and recovery (\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Furthermore, working horses experience markedly elevated sodium and chloride changes compared with maintenance, and these electrolyte losses increase in proportion to workload and ambient temperature (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEffective post-exercise fluid recovery depends on replenishing both water and electrolytes, because electrolyte replacement supports osmotic regulation and improves net fluid retention. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Oral electrolyte solutions are widely utilized in equine sports medicine because they enhance intestinal ion uptake, promote osmotic movement of water into the extracellular fluid compartment, and support restoration of PV (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Electrolyte mixtures can also stimulate voluntary drinking behavior, improving total fluid intake during recovery and contributing to more complete rehydration (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Collectively, these data highlight the importance of electrolyte-based hydration strategies in horses undergoing moderate to intense work resulting in fluid loss through evaporative cooling.\u003c/p\u003e \u003cp\u003eBeyond traditional electrolyte formulations, glycerol has been evaluated as an osmotic agent capable of inducing \u0026ldquo;hyperhydration\u0026rdquo; states in humans and equines. In humans, ingesting glycerol with water increases plasma osmolality, reduces urine output, and expands total body water for several hours, thereby enhancing fluid availability during subsequent exercise (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Similar observations have been reported in horses, where nasogastric administration of glycerol solutions produces transient hyperhydration characterized by increased renal water conservation and greater voluntary water intake (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Together these findings suggest that glycerol can augment oral rehydration strategies by increasing the proportion of ingested water that is retained rather than excreted, potentially contributing to improved cardiovascular and thermoregulatory stability during recovery.\u003c/p\u003e \u003cp\u003eCreatine supplementation represents another strategy to influence fluid distribution and retention as an ergogenic aid. In humans, creatine loading protocols consistently increase muscle creatine content and are associated with increases in total body water and intracellular water (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). A substantial body of literature supports the safety and efficacy of creatine supplementation across athletic and clinical populations, with no evidence of harmful fluid shifts when taken at standard doses (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). More recent reviews emphasize creatine\u0026rsquo;s potential role in supporting cellular hydration, though no research has evaluated creatine\u0026rsquo;s hydration-related effects in horses, representing a meaningful gap in equine physiology.\u003c/p\u003e \u003cp\u003eTogether, these findings suggest that horses can experience large fluid and electrolyte losses via sweat, which can challenge re-hydration and recovery. Furthermore, oral electrolyte solutions enhance fluid intake and promote plasma volume restoration, whereas glycerol and creatine may further support fluid retention via osmotic mechanisms and increases in total body and intracellular water content, respectively. However, this combination of ingredients has not been tested as a hydration supplement in equine athletes.\u003c/p\u003e \u003cp\u003eThe purpose of this preliminary investigation was to evaluate the effects of a novel oral electrolyte supplement containing glycerol and creatine on post-exercise fluid consumption, body mass recovery, and hematologic markers of hydration in horses. We hypothesized that, compared with water alone, the electrolyte, glycerol, and creatine supplement would: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) increase voluntary water intake during the post-exercise hydration period, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) enhance water retention as assessed by recovery of body mass, and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) promote greater intravascular fluid expansion.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eSix Quarter Horses (three mares and three castrated males) from a privately managed training facility were enrolled. All horses were deemed healthy based on physical examination by a licensed veterinarian (DVM). Mean baseline body mass was 506 kg (range 463\u0026ndash;566 kg). Horses were maintained under consistent husbandry practices, including a routine morning grain meal prior to each day\u0026rsquo;s data collection. Horses also received a half flake of alfalfa hay during the 3-hour hydration period on both study days. As all procedures consisted of routine exercise, standard handling, and non-invasive jugular venipuncture performed by a veterinarian as part of normal clinical monitoring, Institutional Animal Care and Use Committee (IACUC) approval was not required. This was consistent with American Veterinary Medical Association guidelines for observational studies conducted within the scope of ordinary veterinary care. Owner consent was obtained for participation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy Design\u003c/h3\u003e\n\u003cp\u003eA crossover, repeated-measures design was used, with each horse completing two experimental conditions on consecutive days. On Day 1 (water condition), horses were exercised and then provided free-choice access to plain water during a 3-hour hydration period. On Day 2 (electrolyte condition), horses followed the same protocol except they received an oral electrolyte paste supplement (100X Equine, HydraMax\u0026trade;) containing electrolytes, creatine, and glycerol, administered at the start of the hydration window. Blood sampling, body mass measurements, and water intake assessments were performed in the same order and at equivalent timepoints relative to exercise on both days. All data were collected in the morning following normal feeding to minimize diurnal variation. Data collection occurred between 10:00 a.m. and 3:30 p.m. Ambient temperature ranged from 20.6\u0026ndash;31.1\u0026deg;C with mean relative humidity of approximately 67%, and conditions were consistent across study days.\u003c/p\u003e\n\u003ch3\u003eExercise Protocol\u003c/h3\u003e\n\u003cp\u003eEach horse completed a standardized 20-minute lunge line exercise session designed to elicit a moderate cardiovascular and musculoskeletal workload, consistent with typical training. The protocol consisted of 10 minutes of trotting (5 minutes each direction), followed by 10 minutes of loping (5 minutes each direction). Horses were not tacked and were exercised in a halter attached to a lunge line in a covered outdoor arena with sand\u0026ndash;fiber footing. Following exercise, horses completed a 30-minute cool-down period consisting of hand walking and standing rest prior to post-exercise measurements.\u003c/p\u003e\n\u003ch3\u003eTreatment Conditions\u003c/h3\u003e\n\u003cp\u003eDuring the water condition, horses had ad libitum access to plain water for 3 hours, provided in two buckets. Each bucket contained 14.4 kg of water (28.8 kg total available). No supplement was administered. During the electrolyte condition, horses received one full serving of the electrolyte paste supplement according to manufacturer instructions immediately prior to ad libitum access to plain water for 3 hours, provided in the same manner as the water condition.\u003c/p\u003e\n\u003ch3\u003eBlood Sampling and Analysis\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected at five timepoints relative to exercise: baseline (pre-exercise), immediately following the cool-down period (Post-Exercise), and at 1, 2, and 3 hours of hydration (1-Hour, 2-Hour, and 3-Hour). Samples were obtained via jugular venipuncture using sterile technique and collected into EDTA tubes, then refrigerated until analysis. All samples were processed within 24 hours at an Antech Diagnostics Veterinary Laboratory. Measured variables included hematocrit, hemoglobin, total plasma protein, and fibrinogen. Percent change in plasma volume (PV) was calculated using the Dill and Costill Eq.\u0026nbsp;(18).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBody Mass and Water Intake\u003c/h2\u003e \u003cp\u003eBody mass was measured at baseline, post-exercise, and at the end of the 3-hour hydration period using a platform livestock scale (Model 244244, Global Industrial, Port Washington, NY, USA; capacity 1000 kg; readability 0.45 kg). The scale was calibrated by the user according to manufacturer instructions prior to data collection. Horses were weighed wearing a halter only, and consistent positioning was maintained across measurements. Water buckets were weighed immediately before and after the hydration period using a portable medical scale (Model SF-891, VEVOR, Kent, WA, USA; capacity 180 kg; readability\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 kg). Water intake was calculated as the difference between pre- and post-hydration bucket weights, assuming a density of 1.0 kg/L.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStatistical Design\u003c/h3\u003e\n\u003cp\u003eA two-way repeated-measures analysis of variance (ANOVA) was used to evaluate the effects of Condition (Water, Electrolyte), Time, and their interaction on hematologic variables (hematocrit, hemoglobin, total protein, and fibrinogen) and on body mass. For hematologic outcomes, three timepoints were analyzed: baseline, post-exercise, and the mean of the 1-, 2-, and 3-hour post-hydration samples. Percent change in plasma volume (ΔPV) could only be calculated for the post-exercise and post-hydration timepoints; therefore, a 2 \u0026times; 2 repeated-measures ANOVA was used for ΔPV, with Condition and Time as within-subject factors. When a significant Condition x Time interaction was detected, Bonferroni-adjusted paired post hoc comparisons were performed to identify differences between conditions and/or timepoints. Water intake was measured once per condition and compared using paired-samples t-tests. All analyses were performed within subject, with horse treated as the repeated factor. Statistical analyses were conducted using GraphPad Prism (Version 10).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHematologic Measures\u003c/h2\u003e \u003cp\u003eAll hematological data are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Baseline hematocrit did not differ between the water and electrolyte conditions (p\u0026thinsp;=\u0026thinsp;0.31). Hematocrit changed significantly over time (p\u0026thinsp;=\u0026thinsp;0.0014), with no main effect of condition (p\u0026thinsp;=\u0026thinsp;0.72) and no Condition x Time interaction (p\u0026thinsp;=\u0026thinsp;0.18). The baseline to post-exercise change did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.11). Baseline hemoglobin did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.30). Hemoglobin demonstrated a significant time effect (p\u0026thinsp;=\u0026thinsp;0.003), with no main effect of condition (p\u0026thinsp;=\u0026thinsp;0.47) and no Condition x Time interaction (p\u0026thinsp;=\u0026thinsp;0.41). The baseline-to-post-exercise change did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.27). Baseline plasma protein did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.19). Plasma protein post-exercise differed between conditions (p\u0026thinsp;=\u0026thinsp;0.00016) and changed over time (p\u0026thinsp;=\u0026thinsp;0.0061), with no Condition x Time interaction (p\u0026thinsp;=\u0026thinsp;0.26). The baseline-to-post-exercise change did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.16). Baseline fibrinogen did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.27). Fibrinogen did not differ by condition (p\u0026thinsp;=\u0026thinsp;0.70), time (p\u0026thinsp;=\u0026thinsp;0.94), or Condition x Time interaction (p\u0026thinsp;=\u0026thinsp;0.28). The baseline-to-post-exercise change did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.26). Calculated percent change in plasma volume (ΔPV) differed between conditions (p\u0026thinsp;=\u0026thinsp;0.033) and changed over time (p\u0026thinsp;=\u0026thinsp;0.0078), with no Condition x Time interaction (p\u0026thinsp;=\u0026thinsp;0.99) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mean ΔPV during the post-hydration period did not differ significantly between conditions (p\u0026thinsp;=\u0026thinsp;0.095).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematologic values across time and stratified by condition.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCondition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHct (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003eHgb (g/dL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasma Protein (g/dL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003eFibrinogen (mg/dL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eΔPV (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBaseline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 (35.0\u0026ndash;44.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 (11.2\u0026ndash;14.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (6.2\u0026ndash;7.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e158.0\u0026thinsp;\u0026plusmn;\u0026thinsp;11.9 (127.4\u0026ndash;188.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElectrolytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 (35.1\u0026ndash;46.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 (11.3\u0026ndash;15.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 (6.1\u0026ndash;6.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e152.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1 (121.3\u0026ndash;183.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePost-Exercise\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 (37.4\u0026ndash;44.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 (12.2\u0026ndash;14.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 (6.2\u0026ndash;7.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e153.5\u0026thinsp;\u0026plusmn;\u0026thinsp;10.5 (126.5\u0026ndash;180.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u0026ndash;3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 (\u0026ndash;14.9\u0026ndash;7.3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElectrolytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 (36.8\u0026ndash;44.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 (11.8\u0026ndash;15.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (6.0\u0026ndash;6.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e159.3\u0026thinsp;\u0026plusmn;\u0026thinsp;15.4 (119.7\u0026ndash;199.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003cp\u003e(\u0026ndash;9.8\u0026ndash;9.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHydration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 (34.6\u0026ndash;38.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 (11.3\u0026ndash;12.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (6.2\u0026ndash;6.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e159.7\u0026thinsp;\u0026plusmn;\u0026thinsp;7.3 (144.3\u0026ndash;175.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 (10.5\u0026ndash;18.8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElectrolytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 (34.3\u0026ndash;38.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 (11.4\u0026ndash;12.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 (6.0\u0026ndash;6.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c10\" namest=\"c8\"\u003e \u003cp\u003e151.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5 (135.8\u0026ndash;167.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 (12.4\u0026ndash;24.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eStatistical Effects\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003e\u0026dagger;\u0026Dagger;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003e\u0026dagger;\u0026Dagger;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e \u003cp\u003eHematocrit (Hct), hemoglobin (Hgb), plasma protein, fibrinogen and percent change in plasma volume (ΔPV) across Baseline, Post-Exercise, and Hydration periods for Water and Electrolyte conditions. Values presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (95% confidence intervals). Hydration values represent the average of the 1-, 2-, and 3-hour post-exercise hydration timepoints. \u003csup\u003e\u0026dagger;\u003c/sup\u003eSignificant main effect of Time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); \u003csup\u003e\u0026Dagger;\u003c/sup\u003eSignificant main effect of Condition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e**Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Approximately here**\u003c/p\u003e \u003cp\u003e**Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Approximately here**\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBody Mass and Water Intake\u003c/h2\u003e \u003cp\u003eBaseline body mass did not differ between the water and electrolyte conditions (p\u0026thinsp;=\u0026thinsp;0.74), and post-exercise mass also did not differ between conditions (p\u0026thinsp;=\u0026thinsp;0.54). A significant Condition x Time interaction was detected for body mass (p\u0026thinsp;=\u0026thinsp;0.004), and a time effect was observed (p\u0026thinsp;=\u0026thinsp;0.043). At the end of the hydration period, body mass differed between conditions (p\u0026thinsp;=\u0026thinsp;0.0219) demonstrating that the supplemented condition resulted in a greater preservation of body mass than water alone. Voluntary water intake during the hydration period was significantly greater in supplemented horses than in the water condition (p\u0026thinsp;=\u0026thinsp;0.0079).\u003c/p\u003e \u003cp\u003e**Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Approximately here**\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e**Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Approximately here**\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e Data from this preliminary study suggest that oral administration of an electrolyte supplement containing glycerol and creatine prior to ad libitum water access may improve post-exercise hydration compared with water alone. Horses receiving the supplement consumed more water, recovered a greater proportion of post-exercise body mass, and exhibited greater calculated PV values at each timepoint.\u003c/p\u003e \u003cp\u003eElectrolyte supplementation is a well-established strategy to replace sweat losses and support hydration in equine athletes. Horses can lose large volumes of hypertonic sweat during exercise, resulting in substantial sodium, chloride, and potassium depletion, altered plasma osmolality, and reduced thirst drive if electrolytes are not replaced adequately (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In the present study, the supplemented condition showed greater increases in PV, which is consistent with improved intravascular fluid delivery. Similar improvements in circulating blood volume, voluntary water intake, and recovery from dehydration have been reported in equines receiving electrolyte-containing formulations following endurance exercise bouts (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The current data suggest that, even following moderate lunge-line exercise, electrolyte replacement combined with glycerol and creatine may enhance post-exercise fluid recovery and help reduce cardiovascular and thermoregulatory strain in subsequent work.\u003c/p\u003e \u003cp\u003eThe addition of glycerol may have acted synergistically with electrolytes to enhance fluid retention and therefore restoration of body mass post-exercise. Glycerol is an osmotic compound that has the capability to increase total body water and reduces renal free-water clearance, thereby prolonging the retention of ingested fluids (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In this preliminary study, horses in the supplemented condition recovered more of their lost body mass than in the water condition, and in several cases approached or exceeded baseline mass by the end of the hydration period. This suggests a net positive water balance with supplementation of the novel electrolyte supplement. Previous research in horses has demonstrated that glycerol containing solutions can induce transient hyperhydration, increase water intake, and enhance renal water conservation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Similarly, human research has shown that glycerol solution ingestion can increase total body water, sustain hyperhydration, and may reduce thermoregulatory and cardiovascular strain during prolonged exercise or heat exposure (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Recent research in runners further confirms that glycerol hyperhydration improves fluid retention and can positively influence thermal responses (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Although urine output was not measured in the present study, the observed pattern of body-mass recovery is consistent with these osmotic and renal effects.\u003c/p\u003e \u003cp\u003eCreatine supplementation may also have contributed to the improved hydration profile, likely through effects on intracellular water. Creatine is taken up into skeletal muscle via a sodium-dependent transporter, increasing intracellular osmolality and drawing water into the cell (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In humans, creatine loading reliably increases total body water and lean mass, with a substantial component of this increase attributed to intracellular water expansion (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Reviews and organizational position statements support the safety of creatine supplementation and highlight its consistent effects on cellular hydration and muscle energetics (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Although equine specific data on creatine use are limited, the combination of creatine with electrolytes and glycerol in the present formulation may have provided an additional stimulus for water retention. Given that both body mass and calculated PV improved beyond water alone, the overall hydration response is consistent with a supplement acting across multiple fluid compartments, even if the precise intracellular contribution in horses remains to be determined.\u003c/p\u003e \u003cp\u003eSubstantial individual variability was evident in the hydration response to electrolyte supplementation. One horse in particular displayed a relatively larger improvement during the electrolyte condition than the group mean, indicating an augmented physiological sensitivity to osmotic and electrolyte driven hydration mechanisms. During the hydration period, water intake was nearly twofold greater with electrolyte supplementation (14.8 kg consumed) compared to water alone (7.6 kg consumed). This intake was accompanied by an approximately threefold greater recovery of body mass relative to the water condition (+\u0026thinsp;6.5 kg electrolyte vs. \u0026minus;\u0026thinsp;3.5 kg water). Plasma volume expansion was also 1.5-fold greater with supplementation, suggesting enhanced intravascular rehydration compared to water alone. These augmented responses in a single horse suggest that some horses possess an inherently stronger drive to drink or more pronounced renal and cardiovascular sensitivity to osmotic changes, leading to a more robust fluid retention profile when electrolytes and osmolytes are provided. Such inter-individual variation has been reported in equine hydration research and likely reflects differences in sweat losses, thirst perception, renal concentrating capacity, and overall fluid-regulatory physiology (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The present findings emphasize that certain horses may derive markedly greater benefit from electrolyte containing supplements, reinforcing the need to consider individual responsiveness when developing hydration strategies.\u003c/p\u003e \u003cp\u003eThe present results are broadly consistent with prior work demonstrating that formulations containing electrolytes alone or in combination with glycerol can enhance water intake, support PV recovery and reduce indices of dehydration in horses (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). What distinguishes this preliminary study is the inclusion of creatine and glycerol within an equine targeted supplement and the focus upon moderate intensity, routine exercise rather than only high-intensity or long duration endurance workloads. Many performance horses perform repeated, submaximal sessions rather than prolonged competition efforts. The initial finding that water alone did not fully restore hydration status, whereas the supplement did, is practically relevant for daily training management. By improving both fluid intake and retention, such supplementation may help maintain more stable hydration across training days and reduce the cumulative strain of repeated mild-to-moderate dehydration. Additionally, no adverse effects of this electrolyte, glycerol, and creatine supplement were observed during the study, nor upon the subsequent days.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. First, fluid compartments were not directly measured. Thus, intracellular versus extracellular water distribution cannot be distinguished, and conclusions about compartment specific shifts remain inferential. Second, urine volume and urinary electrolyte excretion were not assessed, which limits interpretation of renal responses to glycerol and electrolytes. Third, the sample size (n\u0026thinsp;=\u0026thinsp;6) is modest for a full research study, and may limit the detection of smaller effects. Finally, the fixed order of conditions (water day followed by supplement day) introduces potential sequence effects, although the within-subject design reduces inter-individual variability and strengthens internal validity.\u003c/p\u003e \u003cp\u003eIn summary, an electrolyte, glycerol, and creatine supplement administered prior to free water access enhanced post-exercise rehydration compared with water alone in moderately exercised horses. The supplement increased voluntary drinking, improved body-mass recovery, and produced greater calculated PV expansion, indicating more effective hydration. These preliminary findings support the use of combined electrolyte, creatine, and glycerol formula to improve post-exercise fluid recovery in equine athletes and justify further research to validate these initial findings. Further research studies are planned to address the limitations of the current preliminary study and to assess the relative efficacy of electrolytes alone compared to electrolytes with the addition of creatine and glycerol. Furthermore, research is planned to inform the development of guidelines for hydration management of horses exercising or training consistently in hot and humid environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor Contributions: Conceptualization, R.P. and J.D.; methodology, R.P.; formal analysis, R.P.; investigation, R.P.; writing: original draft preparation, R.P.; writing: review and editing, J.D.; supervision, J.D. All authors have read and agreed to the published version of the manuscript. Contributing author: Dr. Jacquelyn Dietrich [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical review and approval were not required for this study given that all procedures were performed by a licensed veterinarian during standard training and monitoring.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOwner consent was obtained prior to horse enrollment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no external funding. The electrolyte supplement used in this study was provided by 100X Equine. All remaining study costs were covered by the participating horse owners.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request due to patient/owner privacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge Abigail Nesbit and Megan Thelen for their significant contributions to this project. Their commitment to horse safety and their attention to detail greatly enhanced the quality of data collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest. 100X Equine provided the electrolyte supplement used in this study but had no role in study design, data collection, data analysis, manuscript preparation, or the decision to publish.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFlaminio MJ, Rush BR. Fluid and electrolyte balance in endurance horses. Vet Clin North Am Equine Pract. 1998 Apr;14(1):147\u0026ndash;58. \u003c/li\u003e\n\u003cli\u003eLindinger MI. Oral Electrolyte and Water Supplementation in Horses. Vet Sci. 2022 Nov 10;9(11):626. \u003c/li\u003e\n\u003cli\u003eMcCutcheon LJ, Geor RJ, Hare MJ, Ecker GL, Lindinger MI. Sweating rate and sweat composition during exercise and recovery in ambient heat and humidity. Equine Vet J Suppl. 1995 Nov;(20):153\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eKerr MG, Snow DH. Composition of sweat of the horse during prolonged epinephrine (adrenaline) infusion, heat exposure, and exercise. Am J Vet Res. 1983 Aug;44(8):1571\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eKingston JK, Geor RJ, McCutcheon LJ. Rate and composition of sweat fluid losses are unaltered by hypohydration during prolonged exercise in horses. Journal of Applied Physiology. 1997 Oct;83(4):1133\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eNaylor JR, Bayly WM, Gollnick PD, Brengelmann GL, Hodgson DR. Effects of dehydration on thermoregulatory responses of horses during low-intensity exercise. J Appl Physiol (1985). 1993 Aug;75(2):994\u0026ndash;1001. \u003c/li\u003e\n\u003cli\u003eGeor RJ, McCutcheon LJ. Hydration effects on physiological strain of horses during exercise-heat stress. J Appl Physiol (1985). 1998 Jun;84(6):2042\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eMaier I, Kienzle E. A Meta-Analysis on Quantitative Sodium, Potassium and Chloride Metabolism in Horses and Ponies. Animals (Basel). 2025 Jan 13;15(2):191. \u003c/li\u003e\n\u003cli\u003eMonreal L, Garz\u0026oacute;n N, Espada Y, Ru\u0026iacute;z-Gopegui R, Homedes J. Electrolyte vs. glucose-electrolyte isotonic solutions for oral rehydration therapy in horses. Equine Vet J Suppl. 1999 Jul;(30):425\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eCoenen M. Exercise and stress: impact on adaptive processes involving water and electrolytes. Livestock Production Science. 2005 Feb 1;92(2):131\u0026ndash;45. \u003c/li\u003e\n\u003cli\u003eSampieri F, Schott HC, Hinchcliff KW, Geor RJ, Jose-Cunilleras E. Effects of oral electrolyte supplementation on endurance horses competing in 80 km rides. Equine Vet J Suppl. 2006 Aug;(36):19\u0026ndash;26. \u003c/li\u003e\n\u003cli\u003eD\u0026uuml;sterdieck KF, Schott HC, Eberhart SW, Woody KA, Coenen M. Electrolyte and glycerol supplementation improve water intake by horses performing a simulated 60 km endurance ride. Equine Vet J Suppl. 1999 Jul;(30):418\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eRiedesel ML, Allen DY, Peake GT, Al-Qattan K. Hyperhydration with glycerol solutions. J Appl Physiol (1985). 1987 Dec;63(6):2262\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eWagner DR. Hyperhydrating with glycerol: implications for athletic performance. J Am Diet Assoc. 1999 Feb;99(2):207\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eSchott HC, Patterson KS, Eberhart SW. Glycerol hyperhydration in resting horses. Vet J. 2001 Mar;161(2):194\u0026ndash;204. \u003c/li\u003e\n\u003cli\u003ePowers ME, Arnold BL, Weltman AL, Perrin DH, Mistry D, Kahler DM, et al. Creatine Supplementation Increases Total Body Water Without Altering Fluid Distribution. J Athl Train. 2003 Mar;38(1):44\u0026ndash;50. \u003c/li\u003e\n\u003cli\u003eAntonio J, Candow DG, Forbes SC, Gualano B, Jagim AR, Kreider RB, et al. Common questions and misconceptions about creatine supplementation: what does the scientific evidence really show? J Int Soc Sports Nutr. 2021 Feb 8;18(1):13. \u003c/li\u003e\n\u003cli\u003eDill DB, Costill DL. Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration. J Appl Physiol. 1974 Aug 1;37(2):247\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eFriend TH. Dehydration, stress, and water consumption of horses during long-distance commercial transport. J Anim Sci. 2000 Oct;78(10):2568\u0026ndash;80. \u003c/li\u003e\n\u003cli\u003evan Rosendal SP, Osborne MA, Fassett RG, Coombes JS. Guidelines for glycerol use in hyperhydration and rehydration associated with exercise. Sports Med. 2010 Feb 1;40(2):113\u0026ndash;29. \u003c/li\u003e\n\u003cli\u003eKoenigsberg PS, Martin KK, Hlava HR, Riedesel ML. Sustained hyperhydration with glycerol ingestion. Life Sci. 1995;57(7):645\u0026ndash;53. \u003c/li\u003e\n\u003cli\u003ePatlar S, Yal\u0026ccedil;in H, Boyali E. The effect of glycerol supplements on aerobic and anaerobic performance of athletes and sedentary subjects. J Hum Kinet. 2012 Oct;34:69\u0026ndash;79. \u003c/li\u003e\n\u003cli\u003eHerrera-Amante CA, Garc\u0026iacute;a-Zepeda G, Garc\u0026iacute;a-Zepeda CE, Y\u0026aacute;\u0026ntilde;ez-Sep\u0026uacute;lveda R, Clemente-Su\u0026aacute;rez VJ, L\u0026oacute;pez-Gil JF, et al. Effects of glycerol hyperhidration on the running economy of long-distance runners: a randomized crossover clinical trial. Front Nutr. 2025;12:1630462. \u003c/li\u003e\n\u003cli\u003evan Loon LJC, Oosterlaar AM, Hartgens F, Hesselink MKC, Snow RJ, Wagenmakers AJM. Effects of creatine loading and prolonged creatine supplementation on body composition, fuel selection, sprint and endurance performance in humans. Clin Sci (Lond). 2003 Feb;104(2):153\u0026ndash;62. \u003c/li\u003e\n\u003cli\u003eBuford TW, Kreider RB, Stout JR, Greenwood M, Campbell B, Spano M, et al. International Society of Sports Nutrition position stand: creatine supplementation and exercise. J Int Soc Sports Nutr. 2007 Aug 30;4:6. \u003c/li\u003e\n\u003cli\u003eKreider RB, Kalman DS, Antonio J, Ziegenfuss TN, Wildman R, Collins R, et al. International Society of Sports Nutrition position stand: safety and efficacy of creatine supplementation in exercise, sport, and medicine. J Int Soc Sports Nutr. 2017;14:18.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Horse, electrolytes, hydration, creatine, glycerol","lastPublishedDoi":"10.21203/rs.3.rs-8882565/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8882565/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElectrolyte supplementation is utilized to support hydration in horses. However, limited data are available on formulations that include osmotically active compounds such as glycerol and creatine. These additional ingredients may enhance fluid retention, yet their physiologic effects in equine athletes have not been evaluated. This preliminary study assessed hematologic responses, body mass recovery, and voluntary water intake following moderate exercise with and without supplementation using a commercially available hydration supplement containing electrolytes, creatine, and glycerol. Six healthy quarter horses completed two conditions in a cross-over design: a water-only condition and a supplemented condition. Each study day consisted of a uniform moderate exercise regimen, a recovery period, and a 3-hour hydration period. Hematocrit and hemoglobin decreased over time in both conditions, while total protein concentrations were consistently lower in the supplemented condition. Calculated plasma volume (PV) increased to a greater extent with supplementation (p=0.033). Body mass was preserved during the hydration period in the supplemented condition but declined in the water condition (p = 0.004). Horses also voluntarily consumed more water when supplemented (p = 0.0079). These preliminary findings suggest that an electrolyte supplement containing glycerol and creatine may augment post-exercise rehydration by promoting increased water intake, supporting PV expansion, and improving body mass recovery in moderately exercised horses.\u003c/p\u003e","manuscriptTitle":"Post Exercise Hydration Responses to an Electrolyte, Glycerol, and Creatine Supplement in Horses: A Preliminary Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-24 09:49:00","doi":"10.21203/rs.3.rs-8882565/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-21T14:44:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T04:22:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-17T04:18:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2026-02-14T21:11:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bdb9bb7b-0343-4e46-8913-2e36c0fbc74d","owner":[],"postedDate":"February 24th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:05:39+00:00","versionOfRecord":{"articleIdentity":"rs-8882565","link":"https://doi.org/10.1186/s12917-026-05493-w","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2026-04-20 15:59:55","publishedOnDateReadable":"April 20th, 2026"},"versionCreatedAt":"2026-02-24 09:49:00","video":"","vorDoi":"10.1186/s12917-026-05493-w","vorDoiUrl":"https://doi.org/10.1186/s12917-026-05493-w","workflowStages":[]},"version":"v1","identity":"rs-8882565","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8882565","identity":"rs-8882565","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}