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Monitoring Muscle Health for Improved Performance and Injury Prevention

Alessandro Pezzola

Massimo Ambrosini

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Contents

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Need identification
Biomarkers variations in ISF while training
Performance requirements
State of the art in lactate and pH monitoring
Proposed molecular sensor design and working principle
Foreseen challenges and Urea sensing
Conclusion

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Need Identification: why monitoring muscle health?

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[1,2]

[2]

Optimal training load:

reduces the risk of injuries

improves the efficiency of training

Precise monitoring training load through muscle fatigue is essential for optimal and safe training

[1]: https://metrifit.com/blog/optimizing-load-management/

[2]: https://doi.org/10.1136/bjsports-2015-095788

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Biomarkers

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Lactate and pH show high correlation with muscle fatigue under normal conditions [3,4].
Lactate and pH levels are affected by several pathways [5].

[3]

[4]

ISF offers balance of:

accessibility,
biochemical relevance,
non-invasive monitoring,

essential for wearable diagnostics during sports.

[3]: https://doi.org/10.2165/00007256-200939060-00003

[4]: https://api.semanticscholar.org/CorpusID:56028956

Their combination allows for higher precision in muscle fatigue monitoring

Why ISF?

Why two biomarkers?

[5]: https://doi.org/10.3389/fphys.2024.1376801

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5

[3] : https://doi.org/10.2165/00007256-200939060-00003

[6] : https://doi.org/10.1016/j.talanta.2023.125582

[7] : 10.1111/j.1469-7793.2001.00993.x

[8] : https://doi.org/10.1038/s43246-024-00468-6

pH	Requirements
Sensitivity	59 mV pH⁻¹ [8]
Linear range	6-8 [8]
Sensor lifetime	24 hours
Stability	<1% deviation

Lactate	Requirements
Sensitivity	50 µA mM⁻¹ cm⁻²
Linear range	0.5-10 mM [3]
LOD	10 µM [6]
Sensor lifetime	24 hours
Stability	< 5% deviation

Sensor Requirements

[7]

[3]

Before we can combine them, each sensor must meet specific performance requirements to ensure accurate and reliable monitoring during training sessions.

For pH sensing, the linear range must align with the physiological range of interstitial fluid which goes from 6 to 8 as you can see on the left graphs which shows the variation of pH during 5 min of exercise at several power outputs for 2 different subjects. You can see that the variation occurs within a small range so the sensitivity must be optimal at 59 mV/pH unit and the stability high enough, with deviations kept below 1%, ensuring consistent readings over the sensor's 24-hour lifespan.

For lactate sensing, the right graph represents the physiological range of lactate concentration in blood upon exercise which goes from 0 to 10 mM but in our application we are interested in tracking the anaerobic threshold so we are less restricted on sensitivity and we rely on average state-of-the-art range which is 50 µA/mM/cm². In addition, the LOD must be below 10 µM as it determines the sensor's sensitivity to low concentrations and the extent to which noise influences the accuracy of measurements. Last requirement is to keep the deviations limited to 5% to maintain reliable performance.

What do the three different colors stand for? The power output was 30 W (green), 50 W (blue) and 70 W (red), Exercise was started 5 min after onset of the recording (marked with horizontal bar), 2 plots = 2 different subjects

PH : sensitivity "Most of the pH sensors have a sensitivity of around 59 mV/pH, governed by the Nernst equation, which predicts the theoretical potential change in a chemical reaction."-Nature / linear range from plots (with margin)/ lifetime defined by longest training session we want to monitor/ stability with deviation below 1% bc we want high-precision during the 24h ideally

Lactate : sensitivity defined by the fact that we want to track the lactate threshold which is located after a rather constant slope and before a steep increase in lactate concentration thus should be trackable without the need for a very high sensitivity/ linear range from plot / LOD from average state-of-the-art and small because it determines the influence of noise on the measurements/ Sensor liftime defined by longest training session/ stability with deviation below 5% bc no need to be super precise

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State-of-the-art : Lactate & pH Sensors

[6] : https://doi.org/10.1016/j.talanta.2023.125582

[9] : https://molab.me/continuous-lactate-monitors-for-athletes/

[10] : https://doi.org/10.1021/acs.chemrev.8b00655

Lactate Sensor in Research

PH Sensor in Research

Lactate Sensor on the Market

[6]

[10]

[9]

With these requirements in mind, we turned to the state-of-the-art in lactate and pH sensing to evaluate existing solutions and guide our approach for their combination.

The table on top shows the use of microneedle arrays or single-needles as an efficient methods for lactate monitoring in ISF as it offers precision, non-invasiveness, and compatibility with wearable technologies, making them highly relevant for real-time monitoring during exercise. Lactate sensors for exercise monitoring are even making their way on the market, with companies like IDRO which uses sweat as their medium.

Similarly, potentiometric pH sensing has been widely utilized in research for sweat monitoring and has demonstrated great potential. Building on these achievements, we propose applying these different methods to ISF which as Massimo mentioned presents a more suitable alternative for our application.

[7] Comparison table of the analytical performances of microneedle-based lactate biosensors

[11] Summary of the Representative Works on pH Sweat Monitoring

[10] Processed data from swaet lactate and blood lactate

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Reduced graphene oxide-functionalized polymer microneedle for continuous and wide-range monitoring of lactate in interstitial fluid

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Structure

- Flexible planar microneedle (polyimide base, 125 μm thick).

- 3-electrode system with 0.004686 cm² electrode area:

Working Electrode

- Reduced Graphene Oxide (rGO): High conductivity, large surface area.

- Platinum Nanoparticles (PtNPs): Boost catalytic activity for H₂O₂ oxidation.

- Lactate Oxidase (LOx): Enzyme for lactate detection

3. Nafion Protective Layer

[6] : https://doi.org/10.1016/j.talanta.2023.125582

Working principle:

REZA at al.

The microneedle biosensor works by detecting lactate through an enzymatic reaction. Lactate is converted into pyruvate and hydrogen peroxide by lactate oxidase (LOx). The produced hydrogen peroxide is then oxidized at the sensor’s working electrode, generating a current. This current is proportional to the lactate concentration, allowing us to measure lactate levels in real-time.

- Flexible planar microneedle (polyimide base, 125 μm thick).

- 3-electrode system with 0.004686 cm² electrode area:

- Working Electrode (WE): rGO, PtNPs, and LOx functionalization.

2. Materials

- Reduced Graphene Oxide (rGO): High conductivity, large surface area.

- Platinum Nanoparticles (PtNPs): Boost catalytic activity for H₂O₂ oxidation.

- Lactate Oxidase (LOx): Enzyme for lactate detection (covalently bonded to rGO).

3. Nafion Protective Layer (1.25 wt%)

- Permselective membrane to block electroactive species interference.

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Reduced graphene oxide-functionalized polymer microneedle for continuous and wide-range monitoring of lactate in interstitial fluid

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Parameter	Detail
Sensitivity	30.44 μA mM⁻¹ cm⁻²
Linear Range	0-12 mM
LOD	2.04 μM
Response Time	~8 seconds
Robustness	~91% sensitivity after 6 days of continuous use
Selectivity	Minimal deviation (~2–4%) against glucose, uric acid, acetaminophen.
Reproducibility	RSD < ~5%

Testing:

- Artificial ISF: 43.96 μA mM⁻¹ cm⁻².�- Human Serum: 30.44 μA mM⁻¹ cm⁻² (biofouling effects).

(g)

[6]: https://doi.org/10.1016/j.talanta.2023.125582

RSD= Relative Standard Deviation 🡪 A lower RSD indicates higher precision, meaning the measurements are consistent and close to each other

test of the Au/rGO/PtNPs/LOx/Nafion biosensor over 6 days; (b) effects of interfering substances on the biosensor reading after the addition of 0.3 mM uric acid (UA), 5 mM glucose (Glu), 50 μM ascorbic acid (AA), or 100 μM acetaminophen (AP); (c) reproducibility measurements for 5 microneedles; (d) response time of the fabricated biosensor; (e) chronoamperometric response of the Au/rGO/PtNPs/LOx/Nafion biosensor in artificial ISF; (f) chronoamperometric response of the Au/rGO/PtNPs/LOx/Nafion biosensor in human serum; (g) Determination of the LOD for lactate

Biofouling refers to the accumulation of biological materials, such as proteins, cells, or microorganisms, on the surface of a sensor or other device. In the context of a biosensor like the microneedle sensor, biofouling can have a significant impact on its performance over time.

Reduced Sensitivity: As the biofilm thickens, it can reduce the effectiveness of the biosensor by preventing direct contact between the electrode and the target molecule. This lowers the sensor's ability to detect the analyte accurately

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Wearable microneedle array-based sensor for transdermal monitoring of pH levels in interstitial fluid �

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Microneedle Array (MNA)

-Material: Hybrid polymer (OrmoComp®)

-Microneedle Density: ~10,000 needles/cm²

Two electrodes system:

-WE: Gold-coated microneedles (Au-PMNA) modified with a polyaniline (PA) layer for pH sensing

-RE: Silver chloride (Ag/AgCl)-coated microneedles

Insulating Layer shielding sensor from sweat interference

4. Sensing Principle: Proton-sensitive PA layer undergoes protonation/deprotonation depending on local pH

Enables precise potentiometric measurements with minimal ion interference

[11]: https://doi.org/10.1016/j.bios.2022.114955

Dervisevic et al.

-Needle Dimensions: Height: ~250 μm, Tip: ~2 μm, Base: ~50 μm

Optical images of PMNA-based wearable pH sensor showing a) a two-electrode system with working and reference electrode (scale bar; 4 mm), and b) contact pads made of polyethylene naph- tholate film (PEN) (scale bar; 4 mm)

Sensing Mechanism: The sensor uses a polyaniline (PA) layer that reacts to changes in pH by interacting with hydrogen ions (H⁺).

In acidic conditions (low pH), hydrogen ions protonate the PA layer, meaning they bind to it.
In basic conditions (high pH), the PA layer deprotonates, releasing the hydrogen ions.

Potentiometric Measurement:

The sensor has two electrodes: one is a gold-coated microneedle (working electrode) modified with the PA layer, and the other is a reference electrode (Ag/AgCl).
The electrical potential at the working electrode changes as the PA layer protonates or deprotonates, and this change is measured to determine the pH.

Minimal Interference:

The PA layer is specifically sensitive to hydrogen ions, making the sensor highly selective for pH changes. It avoids interference from other ions like Na⁺, K⁺, or Ca²⁺, ensuring accurate pH measurements.

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Wearable microneedle array-based sensor for transdermal monitoring of pH levels in interstitial fluid �

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[11]: https://doi.org/10.1016/j.bios.2022.114955

Parameter	Detail
Sensitivity	62.9 mV/pH unit
Linear Range	pH 4.00–8.60, R²= 0.9992
Accuracy	±0.036 pH units
Selectivity	High selectivity against interferents (Na⁺, K⁺, Ca²⁺, Mg²⁺) ~0.04 pH units.
Robustness	>15 uses with <10% signal loss
Interference drift	Minimal drift (0.37 mV/hour 0.6% error over 3 hours).

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Combining microneedle sensors into a single design�

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Images made with Biorender.com

Display for readout

Reference

Electrode (pH)

Working

Electrode (pH)

Lactate microneedle

Electronics for signal processing

Working principle : The sensors themselves would be unchanged from the ones provided in literature. The separate chips are only separate for readability reasons and practically all of these operations can be computed on one chip, this means the sensor size will not be much larger due to the electronics.

Specs: (copied from previous sides, just as a reminder if they ask questions on this slide)

Lactate : Single flexible planar microneedle (polyimide base, 125 μm thick).
pH : 10k microneedles of Dimensions: Height: ~250 μm, Tip: ~2 μm, Base: ~50 μm

We plan on having a readout directly on the sensor. This would cause the largest power consumption of the sensor. The computation would not be complicated and as such would only consume negligible amounts of energy. Comparable sensors (Continuous Glucose Monitors) have lifetime of 2 weeks and need similar amounts of in sensor processing.

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Images made with Biorender.com

Enjoy your training

[3]: https://doi.org/10.1016/j.ijpharm.2021.121257

[7] : 10.1111/j.1469-7793.2001.00993.x

Take a break

End of workout

Sensor Readout: Train-Rest-Go home

[3]

[7]

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User calibration : Baseline ISF pH values can vary between 7.35 and 7.45 [1] for different users

Production costs : Material and production costs of sensor are still too high to make this a commercial product

Medium term muscle health : Urea detection can give us a better idea on muscle recovery [15]

Increasing robustness for pathologies : Both Lactate and pH are influenced by pathologies like sepsis and

chronic kidney disorder [14]

[14]: https://www.nature.com/articles/s41392-022-01151-3#Sec11

[15]: https://doi.org/10.1021/acssensors.3c02386

Challenges and future improvements

[13] :https://doi.org/10.1016/j.ijpharm.2021.121257

Challenges:

Further improvements:

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"Combining lactate, pH, and future urea sensing in interstitial fluid offers athletes a cutting-edge tool for optimizing performance, safeguarding muscle health, and unlocking their full potential."

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Bibliography

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[1]: Michael Kenny. “Optimizing Load Management”. Metrifit Ready to Perform, Nov. 1, 2021.

[2]: Gabbett TJ . “The training—injury prevention paradox: should athletes be training smarter and harder?” British Journal of Sports Medicine 2016;50:273-280

[3]: Faude, Oliver, et al. “Lactate Threshold Concepts.” Sports Medicine, vol. 39, no. 6, May 2009, pp. 469–90

[4]: Rosencrans, Adam S. ”The Potential of pH as a Determinant of Muscle Fatigue During Steady-State Exercise”. 2016

[5]: Yang, Geonwoo, et al. “Wearable Device for Continuous Sweat Lactate Monitoring in Sports: A Narrative Review.” Frontiers in Physiology, vol. 15, Apr. 2024

[6] : Reza, S. et al. “Reduced Graphene Oxide-functionalized Polymer Microneedle for Continuous and Wide-range Monitoring of Lactate in Interstitial Fluid.” Talanta, vol. 270, Dec. 2023, p. 125582

[7] : Street, Darrin, et al. “Interstitial pH in Human Skeletal Muscle During and After Dynamic Graded Exercise.” The Journal of Physiology, vol. 537, no. 3, Dec. 2001, pp. 993–98

[8] : Wu, Zixiong, et al. “Interstitial Fluid-based Wearable Biosensors for Minimally Invasive Healthcare and Biomedical Applications.” Communications Materials, vol. 5, no. 1, Mar. 2024

[9] : Vossen, Loek. “Continuous Lactate Monitors for Athletes – Explained.” Molab, Feb. 27, 2024

[10] : Ghoneim, M. T., et al. “Recent Progress in Electrochemical pH-Sensing Materials and Configurations for Biomedical Applications.” Chemical Reviews, vol. 119, no. 8, Mar. 2019, pp. 5248–97

[11]: Dervisevic, Muamer, et al. “Wearable Microneedle Array-based Sensor for Transdermal Monitoring of pH Levels in Interstitial Fluid.” Biosensors and Bioelectronics, vol. 222, Nov. 2022, p. 114955

[12]: Torres-Terán, Iria, et al. “Prediction of Subcutaneous Drug Absorption - Do We Have Reliable Data to Design a Simulated Interstitial Fluid?” International Journal of Pharmaceutics, vol. 610, Nov. 2021, p. 121257

[13]: Li, Xiaolu, et al. “Lactate Metabolism in Human Health and Disease.” Signal Transduction and Targeted Therapy, vol. 7, no. 1, Sept. 2022 [14]: Dervisevic, Muamer, Maximiliano Jesus Jara Fornerod, et al. “Wearable Microneedle Patch for Transdermal Electrochemical Monitoring of Urea in Interstitial Fluid.” ACS Sensors, vol. 9, no. 2, Jan. 2024, pp. 932–41

[14]: Li, X., Yang, Y., Zhang, B. et al. Lactate metabolism in human health and disease. Sig Transduct Target Ther 7, 305 (2022). https://doi.org/10.1038/s41392-022-01151-3

[15]: Dervisevic et al. Wearable Microneedle Patch for Transdermal Electrochemical Monitoring of Urea in Interstitial Fluid, https://pubs.acs.org/doi/10.1021/acssensors.3c02386

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Wearable microneedle array-based sensor for transdermal monitoring of pH levels in interstitial fluid �

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https://doi.org/10.1016/j.bios.2022.114955

a) Photograph of on-body pH measurement in euthanized mouse with zoom-in section showing working and reference electrodes. b) Potentiometric signal recorded during the application of the wearable patch on mouse skin with magnified sections representing the signal (i) before injection of aISF, (ii) after injection of aISF at pH 7.00, and (iii) aISF at pH 7.60. c) pH versus time plot derived from potentiometric signal shown in panel “bˮ.

a) Reproduc- ibility study of 10 freshly prepared wearable patches. b) Operational stability study of the wearable patch by 15 consecutive usages of monitoring pH change in aISF. c) Long-term stability of the wearable patch over 20 days. d) Effect of sterilization processes, UV or 80% ethanol, on patch performance in monitoring pH levels. Error bars represent SD of n = 3.

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Biological pathways influencing lactate and pH�

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respiratory pathway: increased CO₂ in ISF lowers pH, while ventilation raises CO₂, raising pH
renal pathway: the kidneys reabsorb HCO₃ and excrete H⁺ indirectly influencing ISF pH in longer time scales.
tissue damage: releases intracellular contents as H⁺and lactate into the ISF.
inflammation and immune activity: activated immune cells (macrophages) rely on glycolysis, increasing lactate output and acidifying ISF.
hormonal regulation: catecholamines, glucagon and cortisol can increase glycolysis during stress, leading to elevated lactate levels.

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How and When lactate forms

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During high metabolic activity, cells rapidly break down glucose through glycolysis to produce ATP. When oxygen is limited or the energy demand exceeds oxidative capacity, pyruvate builds up and is converted to lactate by the enzyme lactate dehydrogenase (LDH). This process regenerates NAD⁺, allowing glycolysis to continue. Lactate is then either used as an energy source in other tissues or recycled into glucose, helping sustain energy production despite low oxygen availability.

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pH levels and high-intensity activities

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During normal, low-intensity activities, the breakdown of ATP during muscle contraction releases H⁺, but bicarbonate buffer system in the ISF manages to neutralize excess H⁺. But, during intense activities, the production of H⁺ outpaces the buffer capacity, leading to a further acidification.

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Why 59 mV/pH sensitivity?

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The sensitivity of 59 mV/pH is chosen because it closely aligns with the theoretical Nernstian response, providing high precision and reliability for pH measurement. It is optimal for monitoring the narrow physiological pH range while maintaining accuracy and linearity.

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Nafion Protective Layer

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The Nafion layer is a permselective membrane that plays a crucial role in improving the accuracy and reliability of the sensor by minimizing interference:

Selective Permeability:

Nafion selectively allows small, neutral molecules (like lactate and its byproducts) to reach the electrode while blocking larger or negatively charged electroactive species (e.g., ascorbate, urate, chloride ions). This prevents interference from these species in the lactate measurement.

Enhanced Signal-to-Noise Ratio
Hydrophobic and Hydrophilic Properties

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Improving Sensitivity

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Increase Enzyme Loading:

Improve the immobilization of LOx on the electrode by using advanced techniques such as nanostructured carriers (e.g., nanotubes or nanofibers) to increase the enzyme's active surface area.

Optimize Electrode Surface Area:

Use advanced nanomaterials like 3D graphene structures or hierarchical nanoparticle coatings to further increase the electrode's effective surface area for reactions.

Enhance Catalytic Materials:

Incorporate additional catalysts, such as gold nanoparticles (AuNPs) or bimetallic alloys, which can complement PtNPs and improve the oxidation of H₂O₂.

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Biofouling Effect

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Biofouling refers to the accumulation of biological materials, such as proteins, cells, or microorganisms, on the surface of a sensor or other device. In the context of a biosensor like the microneedle sensor, biofouling can have a significant impact on its performance over time.

Reduced Sensitivity: As the biofilm thickens, it can reduce the effectiveness of the biosensor by preventing direct contact between the electrode and the target molecule. This lowers the sensor's ability to detect the analyte accurately

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pH sensing: Why hybrid polymers?

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Microneedles are often fabricated from hybrid polymers because these materials combine mechanical strength, biocompatibility, and flexibility—key requirements for wearable and minimally invasive sensors. The microneedles must be rigid enough to penetrate the skin and access ISF but also flexible enough to avoid breaking under stress. Hybrid polymers offer an optimal balance of stiffness and flexibility. Hybrid polymers are compatible with precise fabrication techniques, such as photolithography and molding, allowing for the production of microneedles with well-defined dimensions. These polymers are naturally insulating, preventing electrical interference between electrodes.

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How can Urea be used to measure Muscle Fatigue?

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Urea is produced in the liver as a byproduct of the breakdown of amino acids, which occurs when proteins in muscle tissue are degraded during exercise. When muscles experience stress or damage (such as during intense exercise), the body breaks down muscle proteins into amino acids, which are used for energy or to rebuild muscle tissue. This process leads to the production of nitrogen, which is then converted into urea and excreted through urine. Elevated urea levels often reflect increased muscle protein breakdown, a common response to exercise, particularly in the recovery phase when muscles are repairing and rebuilding. A slow decrease in urea after exercise could suggest poor recovery, potential overtraining, or inadequate rest and nutrition. On the other hand, a quick return to baseline levels may indicate efficient muscle recovery and an effective balance between protein breakdown and synthesis