This research was implemented in accordance with the requirements of the Declaration of Helsinki and the protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Army Medical University. The clinical trial registration number is ChiCTR2000038673.
All subjects were 18–30 years old and agreed to participate in this study and signed an informed consent. The diagnostic criteria for dry eye and pre-clinical dry eye were as follows: Symptoms and positive result of non-invasive tear breakup time constitute the diagnosis of dry eye. The screening Ocular Surface Disease Index (OSDI) confirmed that a patient might have dry eye and triggered diagnostic testing of non-invasive tear breakup time. Positive result were OSDI score ≥ 13 and average non-invasive tear breakup time (A-NITBUT) < 10s according to the Tear Film and Ocular Surface Society Dry Eye Workshop II (DEWS II) . Symptomatic patients without demonstrable clinical signs were differentiated into pre-clinical dry eye . Subjects were excluded if they had neuropathic pain, allergic conjunctivitis, Sjögren’s syndrome, lacrimal obstruction or other diseases. Subjects were also excluded if they had dry eye treatment or used contact lenses within 1 month, if they had a history of eye trauma or surgery within 1 year or subjects with serious systemic diseases who were not suitable for strenuous exercise.
The test was divided into two parts in order to avoid the possible influences of Schirmer I test on the subsequent measurements of signs associated with dry eye. Each part included 3 groups, namely dry eye without AE group, dry eye with AE group and pre-clinical dry eye with AE group. In part 1, we studied the variations of Schirmer I test and six tear compositions before and at 0, 30 min after AE. The tear compositions tested included dry eye diagnostic factor lactoferrin  and matrix metalloproteinase-9 (MMP-9) , dry eye inflammation marker IL-6 , oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) , (O-acyl)-ω-hydroxy fatty acids (OAHFA) which is closely related to tear film stability , and Mucin 5 subtype AC (MUC5AC) which is considered to be the most abundant secretory mucin in human tears . In part 2, we studied the variations of signs associated with dry eye before and at 10, 20, 40 min after AE including tear meniscus height (TMH), first non-invasive tear breakup time (F-NITBUT), A-NITBUT, tear film lipid layer thickness (LLT), incomplete and complete blinks and partial blink rate (PBR), and the variations of visual acuity before and at 0, 30, 60 min after AE. The number of subjects in each group are reported in Table 1. During the test period, all the subjects were exposed to the same environment, and they were required to fast because eating have an effect on tear secretion .
The venue was outdoors, the ambient temperature was 25–27 °C while the humidity was 50–60% during the test. The measuring time was between 18:00 and 19:00. The AE protocol was defined as jogging for 30 min. According to the 6–20 Rating of Perceived Exertion Scale, the target heart rate was set to 64–76% of the maximum heart rate in order to achieve moderate exercise intensity . The maximum heart rate was defined as 220 minus age .
The Ocular Surface Disease Index (OSDI) questionnaire was used to quantify the subjective symptoms of dry eye . The full questionnaire is available as a supplementary file (online supplementary file 1). The symptoms and environmental triggers for dry eye in the past week were assessed. OSDI score ≥ 13 was considered positive. The score range was 0–100. The higher the score, the more severe the symptoms.
Schirmer I test
The researcher wore gloves and placed the Schirmer test strip (35 mm; DSA Exports, India, without fluorescent agent) at the outer 1/3 of the lateral eyelid margin without using anesthetics. Both eyes were tested simultaneously and the eyes remained closed during the procedure. The strip was removed after 5 min and the length of wetted part up to the indentation line was recorded. Each test was carried out in a quiet and dark environment.
Analysis of tear compositions
Schirmer test strips were used to collect the tears. The test strips were stored in a refrigerator at − 80 °C, and placed in a refrigerator at 2–4 °C overnight before testing. Amount of PBS buffer to be added to each strip was calculated by multiplying the wetted length of the Schirmer test strip (Schirmer test reading + about 5 mm of the head of strip without scale) by 100 μl. After homogenizing, the samples were centrifuged at 2–4 °C for 15 min (2000 rpm). In total, 10 μl of the supernatant was collected for analysis after precipitation. Six compositions of the tear were determined using ELISA kit (Jiangsu Meimian, China). The absorbance was measured at 450 nm with a multifunctional microplate reader (Labsystems Multiskan MS, Finland).
Measurements of signs associated with dry eye
Keratograph 5 M (OCULUS, Wetzlar, Germany) was used to measure TMH, F-NITBUT and A-NITBUT. Images with unclear boundaries of the tear meniscus were deleted to avoid affecting image analysis. The F-NITBUT defined as the time in each measurement between the last complete blink and the first perturbation or irregularity of the rings of the Placido disc reflected on the corneal surface. The A-NITBUT defined as the average of all tear film breakups occurring in the measured period in each measurement. The measurement was performed twice by the same ophthalmologist and the mean was taken. LipiView LVI-1001 (TearScience, Inc., Morrisville, North Carolina) was used to measure LLT and shot 20s video which automatically recorded incomplete and total blinking . Number of complete blinks (number of total blinks minus incomplete blinks) and PBR (number of incomplete blinks / total blinks) were obtained using simple calculation.
ETDRS chart was used to measure the best corrected visual acuity.
Statistical analyses were performed with SPSS version 20.0 software package (IBM Corp., Armonk, NY, USA). Data was expressed as median (25% interquartile, 75% interquartile) or mean ± standard deviation. The Mann-Whitney U test was applied to compare the age and OSDI score between two groups. The Schirmer I test, tear compositions, signs associated with dry eye and visual acuity at different time points were compared using two-way repeated measures analysis of variance. Bonferroni correction was used for multiple comparisons. The level of significance was set at P < 0.05.
The sample size was calculated using PASS software (version 15.0, NCSS, LLC). According to the results of the A-NITBUT in preliminary experiment, the sample size was estimated by adopting the significance level (α) as 0.05, the desired power (1-β) as 0.85, the autocorrelation coefficient as 0.7 and the standard deviation of the population of 5.1. The estimated sample size was at least 29 eyes in each group.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.