Effect of Exercise on Salivary pH, Amylase, Mucin, and Total Protein Concentration of Unstimulated Whole Saliva in Pakistani Cohort

ABSTRACT

Objective:  To assess the effects of physical exercise on salivary pH, amylase, mucin, and total protein concentration.

Material and Methods:  Saliva samples were collected from 34 participants (21 males, 13 females) at four time points: rest (control), moderate-intensity exercise, high-intensity exercise, and recovery (post-30 min rest). Salivary pH was measured using a pH meter, while amylase, mucin, and total protein concentrations were analyzed using ELISA.

Results:  Salivary pH remained stable throughout. Amylase levels significantly increased (p<0.05) post-moderate (males: 85.66 ± 2.79 units/mL; females: 85.46 ± 2.36 units/mL) and high-intensity exercise (males: 104.42 ± 1.91 units/mL; females: 103.69 ± 2.05 units/mL), declining during recovery. Mucin levels also rose significantly (p<0.05) post-moderate (males: 3.99 ± 0.79 mg/mL; females: 3.95 ± 0.73 mg/mL) and high-intensity exercise (males: 4.57 ± 0.68 mg/mL; females: 4.52 ± 1.07 mg/mL), then decreased in recovery. Total protein concentration followed a similar trend, increasing post-moderate (males: 2.80 ± 0.62 mg/mL; females: 3.95 ± 0.73 mg/mL) and high-intensity exercise (males: 3.94 ± 1.04 mg/mL; females: 3.63 ± 0.59 mg/mL), then declining during recovery.

Conclusion:  Moderate and high-intensity exercise significantly increased salivary amylase, mucin, and total protein levels, while salivary pH remained unaffected.

Keywords:
Saliva; Motor Activity; Enzymes.

Introduction

Human saliva is a complex fluid comprising organic and inorganic molecules, nucleic acids, lipids, carbohydrates, and water [¹]. Compositionally, it also includes cellular debris, microbes, and gingival exudate [²]. The three major salivary glands in the human body are parotid, submandibular, and sublingual, providing ∼85% of the total saliva [³,⁴]. The sympathetic innervation stimulates thicker saliva production of low volume and high protein concentration, whereas parasympathetic innervation produces more liquefied saliva of high volume and low protein concentration [⁵]. Moreover, saliva produced by the parotid gland comprises a large amount of amylase (an enzyme that initiates carbohydrate digestion in the mouth). In contrast, the submandibular gland predominantly secretes saliva rich in a glycoprotein called mucin (responsible for lubrication) [⁶].

Total protein constitutes a significant component of saliva, comprising amylase, proline-rich proteins, statherin, and immunoglobulins [⁷]. It has been reported that salivary amylase accounts for about 50% of the total protein; however, the amylase's enzymatic activity and quantity vary significantly among individuals [⁸,⁹]. Salivary amylase cleaves α-1,4-glycosidic bonds of polysaccharides, which initiates the process of digestion of polysaccharides [¹⁰]. Additionally, amylase provides receptors for the adherence of various bacteria and forms a structural element of the pellicle layer, though its functions remain unknown [¹¹,¹²]. Salivary mucin provides adhesive and viscoelastic functions to the saliva [¹³]. Mucin also transports antibacterial salivary proteins throughout the oral cavity, which maintains their levels in the salivary pellicle and protects the salivary proteins against proteolytic degradation [¹⁴].

Each salivary constituent performs several functions and forms a functional network. No single salivary component has a conclusive effect on oral condition. Instead, the individual constituents work in an interconnected network, and saliva functions as a whole unit. Various elements influence clinical research outcomes on salivary analysis [¹⁵]. These include gender and age differences, medication use, oral-dental health, and physical activity [¹⁶]. Exercise alters the protein profile within the saliva, and this change is influenced by its intensity and interval [¹⁷]. The autonomic nervous system (sympathetic, parasympathetic, and enteric divisions) reacts differently to bodily exercise [¹⁸]. While the major glands receive innervation from both sympathetic and parasympathetic nerves, sympathetic nerve signaling dominates during exercise. This alteration in the level of nerve signals can cause a rise in protein expression, even when the salivary secretion is of low volume during exercise [¹⁹]. Though reactions to various stimuli vary between the glands [²⁰], research has revealed that the rise in the level of sympathetic stimulus due to physical exercise may control the levels of proteins produced by various salivary glands [²¹,²²]. Chicharro et al. [²³] suggested that after performing exercise for a short duration, the secretory levels of the total protein increased. Another study revealed that the intensity of exercise influences the levels of salivary proteins, including amylase, mucin, and total protein, with an increase observed beyond the anaerobic level [²⁴]. This increase in the secretory level of salivary proteins can be due to the neuronal regulation of saliva that is influenced by physical exercise [²⁵]. These previous studies suggest that exercise impacts salivary protein levels, highlighting the need for further investigation.

The rationale for this study was to investigate how physical exercise affects salivary pH and the secretion of key proteins, such as amylase and mucin. Understanding these changes can provide insights into the influence of exercise on oral health and salivary function. Additionally, there is a lack of studies in the literature that have evaluated the impact of moderate and high-intensity exercise on the levels of salivary amylase, mucin, and total protein in the UWS of the Pakistani cohort. A previously conducted study has evaluated the levels of salivary amylase only and compared them between normal and schizophrenic individuals. This study had only measured the levels of amylase [²⁶]. Therefore, the present study aimed to assess the influence of varying intensity of physical exercise on salivary pH levels, secretion of salivary proteins (amylase and mucin), and total protein concentration. The study's null hypothesis (Ho) was that there would be no effect of physical exercise on these parameters.

Material and Methods

Ethical Considerations and Sample Size Calculation

The ethical review committee of Bahria University Medical and Dental College, Pakistan, approved the study protocol and review committee (ERC12/2019). The project was performed in accordance with the Declaration of Helsinki. All participants gave written consent to inclusion in the study, and the potential risks were explained. Participants were also informed that they could decline participation during the survey without consequences. This study protocol was adopted from the previous study by Ligtenberg et al. [³²]. The sample size was calculated using the software OpenEpi, Version 3. The statistical conditions were a 5% margin of error, a 95% confidence interval, and an expected prevalence of 50% with a known population of 35. Then, a sample size of 35 participants was considered acceptable; however, one participant left the study, resulting in a sample size of thirty-four participants (N = 34) for the present study.

Study Participants

Participants aged 25-40 years were recruited, and the inclusion criteria consisted of non-smokers with no systemic or oral diseases and no current medication use. The exclusion criteria comprised individuals who were unwilling to participate or did not consent to the study, those with systemic and oral diseases, and individuals taking medications for systemic diseases that could alter their salivary flow rate.

Study Protocol

The saliva collection process was conducted over 11 days. Each day, saliva was collected from three study participants. Participants carried out exercise by running for a specific period of time on a motor-driven treadmill (Inter-Track Multi-Function Treadmill IT-700M DC Motor; Intercheim Co., Cairo, Egypt). Each participant took nearly 1 hour and 15 minutes to complete the study protocol required for saliva collection. The room temperature for the study protocol was 20-23 °C. Saliva collection was carried out at four different time intervals, and the study participants' heart rate was monitored at all intervals. Each participant followed the same protocol, which is described as follows.

• Initial resting period;

• After running for 10 mins at 3.5 km/h on a treadmill (moderate-intensity exercise) at a heart rate of about 130 beats/min, followed by rest for 10 mins.;

• After running for another 15 min, 8.5 km/h on a treadmill (high-intensity workout) at a heart rate of about 178 beats/min.;

• After a resting period of 30 min, post-high-intensity exercise.

UWS Collection

The UWS was collected early in the morning (between 8 am and 11 am). The patients were advised not to eat or drink anything for at least two hours before the saliva collection. Before collecting the saliva, participants were asked to rinse their mouths thoroughly to remove any debris or food particles. The patients were then seated on the dental chair and were asked to allow the UWS to collect in their mouth for 5 minutes without swallowing. The UWS was then transferred into the graduated measuring cylinder. The UWS flow rate (UWSFR) was determined by dividing the amount of saliva expectorated (in mm) by five. The container was immediately placed in an ice box to preserve the samples before being frozen at -80 °C. The samples were analyzed within two months of collection.

Salivary Assessments

The pH of UWS was assessed using a pen-type pH meter (LutronTM PH-223, Glassco Scientific & Analytical Co, Taiwan). The pH meter was dipped in the container for 5 seconds, and the pH reading was recorded in the data collection proforma. The pH meter was calibrated before each use.

For UWS amylase estimation, an enzyme-linked immunosorbent assay (ELISA) was performed using a modified technique as suggested by Gunatillaka et al. [²⁷]. All the UWS samples were thawed and then diluted with normal saline. The buffered starch solution was prepared by melting 0.4 g of soluble starch with the hot solution comprising 30 mM sodium chloride, 70 mM sodium benzoate, and 200 mM disodium hydrogen phosphate. Cooling of the mixture was performed while the pH was tuned to 7.0. One mL of the buffered starch was placed within the tubes and then into a water bath for 5 minutes at 37 °C. Twenty microliters of the diluted saliva sample were placed inside the test tube. These test tubes were placed inside a vortex mixer and further incubated in a water bath at a temperature of 37 °C. Subsequently, 8 mL of distilled water and 1 mL of iodine solution (5 mM) were added to the tubes. The contents were mixed carefully, and 250 mL of the contents was placed in a flat-bottomed ELISA plate reader (Biotech Synergy Limited, Madhya Pradesh, India), and its absorbance was assessed at 660 nm.

UWS mucin was estimated using the Alcian Blue method [²⁸,²⁹]. The saliva samples were placed in the tubes and diluted with distilled water. A solution of Alcian blue was added to a 50 mM sodium acetate buffer, which was incubated at room temperature for 30 min. Subsequently, samples were placed for centrifugation for 20 min, and 1 mL of 95% ethanol was vortexed for about 10 s, and again after 5 min, samples were centrifuged. One part of Aerosol OT and two parts of distilled water were combined, followed by the addition of an equivalent amount of ethyl ether. The UWS samples were centrifuged for 15 min and analyzed on an ELISA plate reader at a wavelength of 605nm.

The method described previously in the literature [³⁰,³¹] was followed for the total protein concentration. A working standard was prepared by adding 0.2 mL of Bovine serum albumin and distilled water to five test tubes. Later, one test tube containing distilled water was used as a blank. Further, 4.5 mL of reagent containing 1 mL of 0.5% CuSO₄.5H₂O in water, 48 mL of 2% Na₂CO₃ in 0.1 N NaOH, and 1 mL of 1% NaK Tartarate in water, were added and incubated for 10 min. Then, 0.5 mL of reagent two, containing one part of Folin Ciocalteu’s phenol reagent and water, was incorporated and incubated for 30 min. The absorbance was analyzed at 660 nm, while the standard graphs were strategized utilizing an ELISA plate reader.

Statistical analysis

The statistical analysis was performed using a statistical program for social sciences (SPSS v.21, IBM Corp., Armonk, NY, USA). ANOVA and Post hoc test (Tukey-Kramer test) were performed to analyze the influence of workout intensity and rest on UWS pH, amylase, mucin, and total protein concentration.

Results

Demographically, from a total of 34 participants, 21 (61.7%) were males, and 41.1% belonged to age group of 25-30 years. The mean weight of the study participants was 69.75 ± 7.1 kg, while the height was 169.30 ± 3.41 cm. The mean BMI for the participants was 24.37 ± 2.75 (Table 1).

Table 1
Demographic details of study participants.

Variables	N (%)
Gender
Male	21 (61.7)
Female	13 (38.2)
Age
25-30 years	14 (41.1)
31-35 years	9 (26.4)
36-40 years	11 (32.3)
Weight (Kg)	69.75 ± 7.1
Height (cm)	169.30 ± 3.41
BMI (kg/m2)	24.37 ± 2.75

The pH levels remained nearly constant throughout the different intervals of exercise and at rest (p=0.696) (Table 2).

Table 2
Effect of workout intensity on salivary pH (Mean ± S.D.).

Parameter	Rest	Moderate Intensity Exercise	High-Intensity Exercise	Recovery
pH	7.02 ± 0.16	7.04 ± 0.20	7.06 ± 0.18	7.07 ± 0.18

Levels of Salivary Amylase and Mucin

Salivary amylase levels in males and females initially at rest were 65.56 ± 2.85 units/mL and 64.41 ± 1.75 units/mL, respectively. Amylase levels increased during moderate-intensity exercise for both sexes (males: 85.66 ± 2.79 units/mL, females: 85.46 ± 2.36 units/mL) and were similar to the rest values (p<0.05). During high-intensity exercise, amylase levels increased further for both sexes (males: 104.42 ± 1.91 units/mL, females: 103.69 ± 2.05 units/mL) and were significantly higher compared to the values for rest and moderate-intensity exercise (p<0.05). In the recovery phase, a gradual decline in amylase level was noted for males (75.14 ± 2.24 units/mL) and females (74.23 ± 1.64 units/mL), which proved to be statistically significant (p<0.05) to the moderate and high-intensity exercise values but were not expressive compared to the rest values (p>0.05) (Table 3 and Figure 1).

Table 3
Effect of workout intensity on concentration of different salivary proteins in males and females.

	Workout Intensity
Gender	Rest	Moderate Intensity	High-Intensity	Recovery
	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
Male
Amylase (units/mL)	65.56 ± 2.85a	85.66 ± 2.79bA^	104.42 ±1.91cA$	75.14 ± 2.24a^$
Mucin (mg/mL)	2.77 ± 0.73a	3.99 ± 0.79bA^	4.57 ± 0.68cA$	1.63 ± 0.91a^$
Total Proteins (mg/mL)	1.46 ± 0.28a	2.80 ± 0.62bA^	3.94 ± 1.04cA$	1.57 ± 0.76a^$
Female
Amylase (units/mL)	64.41 ± 1.75a	85.46 ± 2.36bA^	103.69 ± 2.05cA$	74.23 ± 1.64a^$
Mucin (mg/mL)	2.72 ± 0.59a	3.95 ± 0.73bA^	4.52 ± 1.07cA$	1.66 ± 0.55a^$
Total Proteins (mg/mL)	1.47 ± 0.20a	2.73 ± 0.90bA^	3.63 ± 0.59cA$	1.40 ± 0.36a^$

• ANOVA, Post-hoc Tukey test, significance level: p<0.05; Different small superscript alphabets in the same row denote significance; Different capital superscript alphabets in the same row denote significance between moderate and high-intensity exercise;
• ^{^}
  Denotes significance between moderate-intensity exercise and recovery; and
• ^$
  Denotes significance between high-intensity exercise and recovery.

Figure 1
Box plot showing combined salivary amylase levels for males and females after various intervals (rest, moderate-intensity exercise, high-intensity exercise, and recovery).

Salivary mucin levels in males and females at rest were 2.77 ± 0.73 mg/mL and 2.72 ± 0.59 mg/mL, respectively. The levels increased for both sexes after moderate-intensity exercise (males: 3.99 ± 0.79 mg/mL, females: 3.95 ± 0.73 mg/mL) and were significantly different from the resting values (p<0.05). These mucin levels further increased after high-intensity exercise for both sexes (males: 4.57 ± 0.68 mg/mL, females: 4.52 ± 1.07 mg/mL). They were significant compared to the rest and moderate-intensity exercise values (p<0.05). In the recovery phase, a decline in the levels of mucin was observed for males (1.63 ± 0.91 mg/mL) and females (1.66 ± 0.55 mg/mL), which proved to be statistically significant (p<0.05) to the moderate and high-intensity exercise values but were not significant compared to the rest values (p>0.05) (Table 3) (Figure 2).

Figure 2
Box plot showing salivary mucin levels combined for males and females after various intervals (rest, moderate intensity exercise, high intensity exercise, and recovery).

Total Protein Concentration

The concentrations of total salivary protein in males and females at rest were (1.46 ± 0.28 mg/mL) and (1.47 ± 0.20 mg/mL). These levels were increased for both sexes after moderate-intensity exercise (males: 2.80 ± 0.62 mg/mL and females: 3.95 ± 0.73 mg/mL) and were found to be significant (p<0.05) compared to the rest of the values. A further increase was seen in these levels after high-intensity exercise (males: 3.94 ± 1.04 mg/mL, females: 3.63 ± 0.59 mg/mL), and this increase was significant compared to the rest and moderate-intensity exercise values (p<0.05). In the recovery phase, a steady decline in level was seen for males (1.57 ± 0.76 mg/mL) and females (1.40 ± 0.36 mg/mL), which proved to be statistically significant (p>0.05) to the moderate and high-intensity exercise values, but not substantial compared to the rest values (p>0.05) (Table 3) (Figure 3).

Figure 3
Box plot showing the total protein concentration combined for males and females after various intervals (rest, moderate-intensity exercise, high-intensity exercise, and recovery).

Discussion

The current study aimed to investigate the effects of varying-intensity exercise on salivary pH levels, the secretion of salivary proteins (amylase and mucin), and total protein concentration in saliva. Based on the study's findings, the Ho was partially rejected, as a statistically significant change (p<0.05) in the salivary amylase and mucin, and total protein concentration was observed after physical exercise. The Ho was partially accepted as physical exercise did not affect the salivary pH levels. It is known that exercise affects the sympathetic nervous system, which in turn increases the level of salivary mucin, amylase, and total protein content, along with the other salivary components [³³]. In the current research, UWS was used as it has been extensively researched, and stimulation of saliva could alter its protein levels [³⁴].

Regarding salivary pH levels, the normal physiological range of saliva pH values is from 6.2 to 7.6. Even during the resting period, the pH of saliva does not drop below 6.3, as the pH levels are maintained by various salivary components [³⁵]. Results from the current study demonstrated non-significant differences between each time interval, and pH levels of ∼7 were maintained throughout the study. Although a slight increase in the salivary pH levels was observed after exercise, these differences were not significant. Our results are in agreement with a previous study, which also demonstrated that the salivary pH levels do not alter significantly during endurance sports activities [³⁶]. A plausible reason for this finding could be that salivary pH levels do not change immediately during and after exercise; nonetheless, this finding needs further investigation.

Studies have shown that levels of salivary amylase increase when individuals are subjected to pain, stress, and exercise [³⁷,³⁸]. The present study's findings revealed an increase in salivary amylase levels after moderateand high-intensity exercise. The findings align with the outcomes of an earlier study, which demonstrated that the levels of amylase in saliva increase after exercise, and this increase is dependent on the magnitude of the exercise [³⁹,⁴⁰]. Likewise, Flynn et al. [⁴¹] also indicated that moderate-intensity workouts are adequately stressful in increasing salivary amylase levels. The increase in salivary amylase levels observed in the present study after different intensity exercises could be attributed to the fact that exercise stimulates the sympathetic nervous system [⁴²]. Since the concentration of amylase depends on the sympathetic stimulations of the salivary glands [⁴³], exercise could have induced an increase in the levels of amylase observed in the current study.

Furthermore, the present study noted a decrease in salivary amylase levels in the recovery phase. This could be explained by the findings of a previous study, which revealed that exercise or physical activity could be considered a stress factor. Once the stress is removed, there is a decline in salivary amylase levels [⁵].

Mucins provide lubricating properties to saliva that help in deglutition and mastication [⁴⁴]. A rise in mucin levels was observed for each participant during moderate and high-intensity exercise in this study. Similar findings have been reported by Ligtenberg et al. [³²], who also identified an increase in mucin levels following physical exercise. This finding could be attributed to the fact that exercise leads to dehydration, resulting in a decreased salivary rate, which could result in the detection of increased levels of mucins in the saliva [⁴⁵].

Concentrations of total salivary protein were found to be increased after moderate and high-intensity exercise. This surge in total protein levels could be due to the stimulation of the sympathetic nervous system under the influence of exercise [³²]. Additionally, the increased levels of salivary proteins post-exercise may also be attributed to the increased secretion of salivary amylase [⁴⁶], as observed in the present study.

The current study, a single-center cross-sectional study, was conducted on a limited number of participants from Pakistan, all of whom belonged to the 25-40 age group. The younger and older participants were not investigated but can be further evaluated in future studies to determine if similar findings are observed. In addition, only amylase, mucin, and total protein concentration were assessed using ELISA in the present study. Other salivary components must be analyzed in participants to examine their levels of saliva post-exercise. In the present study, the mode of exercise was a treadmill; however, other forms of workouts can also be used, and results can be compared between the different groups in future studies.

Conclusion

The study we conducted provides fresh perspectives on how exercise affects salivary biomarkers by demonstrating that physical activity has a considerable impact on the protein composition of saliva but has no effect on pH. A physiological reaction to exertion has been suggested by the reported rise in salivary amylase, mucins, and total protein content during moderate and high-intensity exercise. After exercise, the subsequent drop to baseline levels suggests a transient effect. These findings contribute to the growing understanding of salivary biomarkers in exercise physiology and highlight the need for further research with larger cohorts to explore additional salivary proteins and their potential clinical relevance.

• Financial Support
  None.

Data Availability

The data used to support the findings of this study can be made available upon request to the corresponding author.

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Edited by

• Academic Editor:
  Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  08 Dec 2025
• Date of issue
  2026

History

• Received
  10 Sept 2024
• Reviewed
  25 Mar 2025
• Accepted
  24 Apr 2025