This prospective, single-masked, randomised controlled clinical trial was conducted at the optometry clinic of Tianjin Medical University Eye Hospital from January 2022 to January 2023.

Myopic children prescribed single-vision lenses were enrolled. The eligibility criteria included aged between 6 and 13 years, exhibiting a spherical equivalent (SE) refraction (post-cycloplegia) from -0.75 to -6.00 D, astigmatism of less than -1.50 D, anisometropia of less than 1.50 D, and a best corrected visual acuity in both eyes of no less than 0.1 LogMAR. Patients were excluded if they had binocular visual abnormalities such as strabismus, any other significant ocular or systemic conditions, had undergone alternative myopia control interventions within the preceding 3 months, or had any condition deemed by an ophthalmologist as a contraindication for participation.

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki, receiving approval from the Ethics Committee of Tianjin Medical University Eye Hospital (Approval No. 2021KY-34), and was registered with the Chinese Clinical Trial Registry (ChiCTR2200055532). Prior to commencement, written informed consent was obtained from all participants and their guardians.

Participants were randomly assigned in a 1:1 ratio to either the once-nightly (QD) or twice-daily (BID) 0.01% atropine group using simple randomisation via a computer-generated random number table, without stratification factors. The participants were not blinded, but the outcome assessors were unaware of the participants' assigned intervention. Those in the QD group received a single drop of 0.01% atropine in each eye, administered nightly, while those in the BID group were treated with a drop in each eye, both morning and evening, with an interval of 10-12 h. The intervention spanned a duration of 1 year. The 0.01% atropine eye drops were supplied by Shenyang Sinqi Pharmaceutical Co., Ltd., with each 0.4 mL containing 0.04 mg of atropine sulphate. The formulation was preservative-free and included excipients such as hypromellose, disodium edetate, sodium dihydrogen phosphate monohydrate, sodium chloride, hydrochloric acid and/or sodium hydroxide, and water for injection. The study coordinators regularly called the legal guardians to inquire about the participants' medication usage to ensure compliance with the regimen.

The primary endpoint was the changes in axial length (AL) after 1 year, with the changes in SE after 1 year serving as the key secondary endpoint. Safety endpoints encompassed near visual acuity (NVA), the amplitude of accommodation (AMP), positive and negative relative accommodation (PRA and NRA), binocular cross cylinder (BCC), monocular and binocular accommodative facility breaks (AFB), photopic and mesopic pupil diameter (PD), intraocular pressure (IOP), first and average non-invasive tear break-up times (NIBUT and NIBUT), tear meniscus height (TMH), redness score (RS), height, and weight. For analytical consistency, data from the right eye of each subject were selected. Follow-ups were scheduled at 3, 6, 9, and 12 months post-initiation of treatment. All follow-ups were completed from 8:00 am to 12:00 am.

Initially, SE was ascertained for each eye independently using an auto-refractometer (KR-800, TOPCON, Japan), with three measurements per eye to derive an average 30 min after cycloplegia with 1% cyclopentolate hydrochloride, one drop each time, with a 10-min interval, for two times. Subsequently, an experienced optometrist conducted a subjective refraction examination using a comprehensive refractometer to finalise the SE recordings. PD was quantified using a pupillometer (SN-M000716 OASIS, USA), averaging three attempts in both illuminated (300-500 Lx) and dim (50-100 Lx) conditions. NVA was measured for one eye at a time using logarithmic visual acuity charts. The process continued until two errors occurred on the same line, with the preceding line's visual acuity value representing the NVA. The AMP was determined by asking the participants to focus on a target, progressively moving closer until blurring was reported. The reciprocal distance from this point to the eye defined the AMP, with three measurements averaged. Additional assessments included PRA and NRA, accommodation lag (BCC), and AFB, using a comprehensive refractometer. AL was measured using the LenSTAR LS-900 (Haag-Streit). Certified optometrists performed three measurements per eye at each visit. If the intra-measurement variation exceeded 0.05 mm, a new set of measurements was taken. The mean of three consistent readings was used for analysis. IOP was determined through a non-contact tonometer (CT-1, TOPCON, Japan), averaging three measurements to ensure accuracy. Using a non-invasive ocular surface analyser (Keratograph 5 M, OCULUS, Germany), tear film stability and volume were assessed through NIBUT and NIBUT, along with TMH and RS.

Accommodative lag was measured using the fused cross-cylinder (FCC) method on a phoropter, following subjective refraction. The participant's distance correction and near PD were first input into the phoropter. A cross-cylinder lens of ±0.50 dioptres was introduced with the negative axis at 90°, and the FCC target was placed at a fixed distance of 40 cm on the near-vision rod. Participants were instructed to keep both eyes open and fixate binocularly on the FCC target under normal ambient lighting conditions. If horizontal lines appeared clearer than vertical ones, this was interpreted as accommodative lag. Positive spherical lenses were added in both eyes until the subject reported equal clarity of the vertical and horizontal lines. The total plus lens power required to achieve this balance was recorded as the accommodative lag.

Information on parental myopia was collected by performing both objective (autorefractor-based) and subjective refraction for each parent, and the SE was calculated to document the presence and degree of myopia.

Drawing upon historical data from vision screenings of primary and secondary school students in Tianjin (unpublished data from the authors), which indicated an average annual AL progression of 0.27 ± 0.22 mm in children aged 5-12 years, we hypothesised no effect with QD administration of 0.01% atropine and a 50% reduction in AL progression with BID administration. Considering a two-sided significance level of alpha =0.1 and a power of 0.8, we determined a requisite sample size of 68 participants. The sample size was increased to 76 subjects to account for an anticipated 10% loss to follow-up, equally divided between the two groups. Sample size calculation was conducted using PASS 15 software.

Continuous data were analysed by the Kolmogorov-Smirnov test for normal distribution using IBM SPSS Statistics 27 and presented mean ± standard deviation (SD). The parameters such as SE, AL, PD, and AMP were analysed at various follow-up intervals before and after medication administration using repeated measures and two-way ANOVA. Independent samples t-tests were used for inter-group comparisons at identical time points. P < 0.05 was considered statistically significant.