With the rise in average life expectancy, skin health and aesthetics have become important factors in quality of life (QOL) [1]. According to a recent study carried out by the American Society of Dermatologic Surgery [2] in 2023, approximately 70% of people said they were considering a cosmetic procedure. “Looking beautiful” is a significant cultural need in contemporary Iranian society, which has one of the highest rates of cosmetic procedures per capita worldwide [3]. Skin lines and wrinkles primarily result from chronic sun exposure, although other factors, such as the loss of subcutaneous fat and exposure to environmental pollutants, also contribute [4].

Although invasive facial rejuvenation methods, such as cosmetic surgery, are effective in creating a youthful appearance, they do little to address the underlying causes of facial aging. Moreover, these methods are expensive and carry their own surgical risks [5]. As a result, less invasive methods, such as injections of botulinum toxin (Botox) and skin fillers, chemical peels, and laser therapy, which are often cheaper and less complicated, are commonly used as supplements or substitutes for surgery [6].

Botox injections and fillers have gained popularity due to their relatively quick effects, minimal invasiveness, and shorter recovery times compared to surgery. However, these procedures have their own side effects, such as eyebrow and lip ptosis or facial asymmetry, and a mask-like appearance in the case of Botox. Filler injections can also lead to local reactions, such as edema, erythema, bruising, and pain, as well as complications related to incorrect injection techniques, including palpable nodules, visible implants, and allergies [7]. To avoid these side effects, simpler and cheaper alternatives, like acupuncture, acupressure, and facial exercises, have become increasingly popular [6,8,9].

Cosmetic acupuncture is a holistic approach to facial rejuvenation that restores the condition of the entire body without injections or surgery. Unlike other methods, it can promote facial rejuvenation by restoring systemic health [8,9]. Acupuncture is generally considered a safe treatment modality, with minimal or no side effects when performed by experienced practitioners [10].

Despite the growing popularity of acupuncture for facial rejuvenation, few randomized clinical trials have evaluated its results on facial wrinkles. Barrett’s [8] U.S. study reported the positive impact of electroacupuncture and Chinese herbal medicine on reducing nasolabial folds in a 26-year-old woman. Donoyama et al. [11] demonstrated that facial acupuncture increased the water and fat content of the skin in two Japanese women (aged 29 and 50), whose skin hydration and fat content were initially lower than average for women of their age. Yun et al. [1] showed that acupuncture improved facial skin elasticity in 27 South Korean women aged 40 to 59. Additionally, a 2017 study conducted by Yun and Choi [12] on 14 South Korean women aged 40 to 59 showed that, although facial acupuncture with thread embedding did not significantly reduce the vertical distance of the nasolabial folds, it did significantly reduce melolabial and nasolabial wrinkles. Finally, Cho et al.’s [13] study demonstrated how the Jae-Seng acupuncture method effectively reduced nasolabial folds and eye wrinkles in 107 South Korean patients aged 20 to 70.

However, despite these findings, no evidence has been found regarding the effects of acupuncture on upper facial wrinkles. Therefore, further research is necessary to explore the impact of acupuncture on rejuvenating the upper facial area. Additionally, while acupuncture is not limited to treating localized points in the affected area, most studies on facial rejuvenation only focus on local facial acupuncture. Few, if any, studies have examined the effects of combining facial and body acupuncture for this purpose. Therefore, we decided to investigate the effects of combined facial and body acupuncture on frown lines in women aged 30-59 who were seeking treatment at the dermatology or acupuncture clinics of Mashhad University of Medical Sciences in Iran.

1. Study design

This study is a parallel randomized waiting-list controlled trial comparing the face and body acupuncture treatment (intervention) group with the waiting-list (control) group in women aged 30-59 years with glabellar frown lines. In total, 72 participants with frown lines will be randomly allocated to the intervention and control groups at a 1:1 ratio. A summarized schedule of the participant timeline and the flow chart of the study procedure are shown in Table 1 and Fig. 1. The study was registered in the Iranian Registry of Clinical Trials on 23 April 2023 (IRCT Id: IRCT20230204057316N1), and participant recruitment began in June 2023.

GLS = glabellar line scale; GAIS = global aesthetic improvement scale; SSS = subject satisfaction scale..

&md=tbl&idx=1' data-target="#file-modal"">Table 1

Schedule of enrolment, interventions, and assessments.

Week	0	0◄──────────────►6	7	12
Study period	Pre-treatment phase	Treatment phase	Post-treatment phase	Follow-up phase
Enrolment	×
Eligibility screen	×
Informed consent	×
Allocation	×
Interventions		×
Treatment group		12 acupuncture sessions (2 sessions per week)
Control group		No treatment
Outcome measurements
Primary outcome
GLS	×		×	×
GAIS			×	×
Secondary outcome
SSS			×	×
SF-36 questionnaire	×		×	×
Photography	×		×	×
Adverse events		×	×	×

GLS = glabellar line scale; GAIS = global aesthetic improvement scale; SSS = subject satisfaction scale..

Figure 1. Flow chart of the study process.

2. Participants

1) Recruitment

Participants will be referred by physicians from outpatient dermatology and acupuncture clinics at Imam Reza Hospital, Mashhad University of Medical Sciences. Additionally, an email will be sent out to university staff to invite volunteers to participate in the trial. A research team member (HH) will then screen the potential participants based on the inclusion and exclusion criteria and will obtain informed consent, including consent for the publication of participant photographs.

2) Sample size

In the absence of a double-arm study in this field, the sample size was calculated based on an effect size of 0.7, derived from a similar previous study [1], an alpha error of 0.05, and a power of 80%. This resulted in 34 participants per group (intervention and control, total = 68). To account for a potential dropout rate of 5%, the final sample size was adjusted to 72 participants.

3) Inclusion and exclusion criteria

The inclusion criteria are: 1) women with frown lines of any severity; 2) Aged 30-59 years; and 3) provided informed consent.

The exclusion criteria are: 1) previous treatment with dermabrasion, deep skin peeling, laser peeling (ablative or non-ablative), botulinum toxin injection, filler injection, or topical steroid treatment in the glabella and forehead area within the past six months; 2) participants with a current or history of skin disease or chronic dermatological condition; 3) pregnancy or lactation; 4) history of herpes; 5) history of skin or metal allergies or needle phobia; 6) history of diabetes or tuberculosis; 7) use of anticoagulant or thrombolytic therapy; and 8) patients with serious systemic conditions (e.g., cardiovascular, pulmonary, renal disease, etc.). After enrollment, any participants who receive additional facial rejuvenation treatments during the study and follow-up phases or miss more than two consecutive or non-consecutive acupuncture sessions will also be excluded.

3. Randomization and allocation concealment

To ensure random allocation, a permuted block method will be used to generate the allocation sequence via Sealed Envelope (http://www.sealedenvelope.com). Participants will receive opaque envelopes with their screening order number (1 to 72) on the outside, and their assigned group (A or B) will be printed on a separate sheet inside. To establish an audit trail, and to maintain allocation concealment and prevent violations, a sheet of carbon paper will be placed on top of the allocation sheet, with the carbon side facing it. The completed insert will then be placed in a blank envelope, with the carbon paper closest to the back of the envelope. Writing the participant’s name and recruitment date on the back of the envelope will transfer this information onto the treatment allocation sheet inside. The allocation sequence will be generated by the trial’s methodologist (MKhR), and the sealed envelopes will be given to a research team member (HH).

4. Blinding

In this study, blinding of the participants or acupuncturists is not feasible. However, outcome assessors, who are independent of the research team, will remain unaware of the participants’ group assignments. The data analyst will be blinded to each participant’s group allocation by using coded labels (A and B) in the datasheet.

5. Interventions

1) Intervention group

The acupuncture treatment protocol for the intervention group was developed in accordance with the Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) [14].

The following points are used in body acupuncture: LIV3 (Taichong), ST36 (Zusanli), SP9 (Yinlingquan), SP10 (Xuehai), LI4 (Hegu), LU7 (Lieque), PC6 (Neiguan), LI11 (Quchi), and DU20 (Baihui) (Fig. 2). Needles (25 mm length × 0.25 mm thickness) are inserted bilaterally. Both the lower limb points (LIV3, ST36, S9, and SP10) and the upper limb points (LI4, LU7, PC6, and LI11) are needled from distal to proximal. Finally, DU20 is needled at the vertex. A total number of 17 needles are used in body acupuncture.

Figure 2. Location of body acupoints.

The facial acupuncture points are BL2 (Zanzhu), Ex-HN4 (Yuyao), TB23 (Sizhukong), and Ex-HN3 (Yintang) (Fig. 3). Bilateral needling is performed using 13 mm (length) × 0.18 mm (thickness) needles. First, Ex-HN3 is needled in the middle of the glabella, between the two eyebrows, followed by BL2, Ex-HN4, and TB23 from medial to lateral along the eyebrow, respectively. A total of seven needles are used for facial acupuncture.

Figure 3. The Corrugator supercilii muscle and location of face acupoints.

For all body and facial acupuncture points, needling is performed first on the right side, then on the left. The depth of needle insertion is determined by the skin thickness and subcutaneous fat at each site, following acupuncture guidelines. All points are needled using the even method.

Finally, intradermal needles (5 mm length × 0.22 mm thickness) are inserted into the depth of the frown lines, with a 2 mm spacing between each needle. Needles are inserted at a 45-degree angle of 45 degrees, with the needle direction upwards, targeting the groove depth.

For the treatment, patients lie in a supine position with a small pillow placed under their head. Needles from Huanqiu are used for all body, face, and frown line acupuncture. The needles remain in place for 20 minutes per session before being removed. If bleeding occurs after removal, cotton wool is applied to the area for 1 min to prevent further bruising. If bruising occurs, Arnica ointment will be applied. In total, 12 treatment sessions will be performed twice a week for six weeks by an acupuncturist (HA) with 14 years of experience. The acupuncture treatment will be completely free for participants in the intervention group. Participants can choose their preferred treatment days and times (morning or afternoon). Participants will receive a text message reminder the evening before their next session.

There is no prohibition for participants except for using any other facial rejuvenation methods during the treatment and follow-up phases, which leads to exclusion from the trial.

2) Control group

The control group will be monitored for six weeks without any intervention and placed on a waiting list. At the end of the study, the group will receive complimentary acupuncture treatment. Selecting an appropriate control method is crucial for ensuring the methodological quality of a randomized controlled trial (RCT). A cross-sectional survey indicated that acupuncture trialists should choose a control method based on the specific aims of their study. However, the survey showed no significant differences between sham acupuncture and a waiting list/no treatment control. Therefore, the authors concluded that a waiting list is an appropriate control method if the aim is to account for the natural progression of disease [15].

6. Outcome measurements

1) Primary outcome

Glabellar Line Scale (GLS): participants in both groups will be photographed at three time points: baseline (week 0), post-treatment phase (week 7), and follow-up phase (week 12).

Photographs of participants’ foreheads will be taken using a Canon 5D Mark III digital camera mounted on a tripod. The photography room will have no windows in order to eliminate outside light. The participant’s chin will rest on a support, and light and other photographic conditions will be standardized according to Honeck et al.’s method [16]. All patients will be photographed at each time point, without makeup, in two different states: at rest and during maximum frown.

The GLS will be evaluated based on photographs taken in both states by three independent physicians. The average score from these three evaluators will be used for each participant. To ensure the validity of the results, clinicians performing GLS assessments will receive GLS training from an expert on our team (MJY). The GLS will be rated for each frown line on a 4-point scale: 0 = no line, 1 = mild, 2 = moderate, and 3 = severe [17]. In cases where a participant has multiple frown lines, the average score will be used.

Global Aesthetic Improvement Scale (GAIS): this evaluation will be performed by a physician independent of the research team. The GAIS assesses the degree of improvement in frown lines based on the GLS during the post-treatment and follow-up phases (weeks 7 and 12) compared to the pre-treatment phase (week 0) for each participant. The GAIS uses a five-point scale: (+2) much improved = improvement of at least two degrees or reaching the zero-degree (no frown line); (+1) improved = improvement of less than two degrees without complete resolution; (0) no change; (–1) worse = worsening of less than two degrees; and (–2) much worse = worsening of at least two degrees [17].

2) Secondary outcomes

The Subject Satisfaction Scale (SSS): this assessment will be completed by the participants themselves at weeks 7 (post-treatment phase) and 12 (follow-up phase) in both groups. The SSS uses a five-point scale for self-evaluation: +2 = very satisfied, +1 = satisfied, 0 = no difference, –1 = unsatisfied, and –2 = very unsatisfied [17].

The Quality of Life (QOL) score: the QOL score will be determined using the SF-36 questionnaire completed by the participants at weeks 0 (pre-treatment phase), 7 (post-treatment phase), and 12 (follow-up phase) in both groups [18].

7. Data management

All data, including adverse events, will be manually recorded immediately after each treatment session by the principal investigator (HA) on a paper sheet numbered with the participant code. Each photograph will be encoded with a three-part code (participant code, photography phase, and the patient’s state at the time of photography) and stored digitally on two separate laptops dedicated to the project. The participants’ QOL and SSS questionnaires, identified by participant code, will be stored in a locked filing cabinet, with restricted access. These records will be kept for at least two years after the publication of the results until a final decision is made by the research vice-chancellor of Mashhad University of Medical Science regarding their final disposition.

8. Confidentiality

The demographic information of the patients will be collected by HH and will be kept with them until the end of the trial. A unique code will be assigned to every participant according to the order of their recruitment, ensuring that their identity remains confidential. Other researchers will only have access to the primary and secondary outcomes of a participant using their code.

9. Statistical analysis

Data analysis will be performed by a statistician who is blinded to the allocation and intervention process, using SPSS statistical software (V.16.0). Descriptive statistics for qualitative and quantitative variables will be presented as frequencies (with percentages) and means (with standard deviations), respectively. For quantitative data comparisons between the two groups, a t-test will be used if the data are normally distributed, with equivalent non-parametric tests used if the data are not normally distributed. ANOVA will be used to compare quantitative variables in cases involving more than two groups. The chi-squared test and Fisher’s exact test will be employed to assess qualitative variables in the two study groups. Paired t-tests will be used for before-and-after comparisons, with the Wilcoxon test used for non-normally distributed data.

The intention-to-treat (ITT) method will be used as the primary analysis method for all efficacy outcomes. The per-protocol (PP) set will be used for sensitivity and consistency analysis to compare results from the ITT set. The PP set will consist of participants who complete the trial without major protocol violations and will provide all outcome measures. The final analysis carried forward will be used to handle missing data for the primary outcome.

If confounders are found to be unequally distributed between the two groups (i.e., if there are statistically significant differences in baseline characteristics), regression models (logistic or linear, depending on the outcome measure) will be used. All tests with be two-tailed, with p < 0.05 denoting statistical significance.

10. Oversight and monitoring

The Trial Management Group (TMG), consisting of members of the trial implementation team (two acupuncturists, a dermatologist, and a methodologist), is led by the principal investigator and is responsible for preparing the protocol and revisions, developing data collection forms, overseeing the day-to-day conduct of the trial, entering data, holding weekly coordination meetings, submitting monthly written reports to the Trial Steering Committee (TSC), and providing quarterly written reports to the Data Monitoring Committee (DMC), following TMC. The TMG is also responsible for publishing the study results.

The TSC, which comprises an independent acupuncturist as chair, an independent dermatologist, an independent methodologist, and the principal investigator, is tasked with reviewing recommendations from the DMC, finalizing the protocol, endorsing TMG actions, providing advice and recommendations to the funder of the trial and the trial team, and reviewing and approving the monthly written TMG reports.

The DMC, composed of members from the research council of Mashhad University of Medical Sciences, is responsible for reviewing safety and efficacy data, providing recommendations to the executive group, and reviewing and approving progress reports.

Finally, audits will be conducted daily by team members, weekly during TMG coordination meetings, and monthly through inspections or by reviewing the TMG’s written reports. These audits will include all steps, such as registration, obtaining informed consent, eligibility and allocation to study groups, adherence to experimental interventions, injury reporting, completeness, accuracy, and timeliness of data collection.

11. Safety considerations and evaluation

As previously mentioned, acupuncture is generally considered a treatment modality with minimal adverse effects. A review by White [19] examined the range and incidence of adverse acupuncture-related events across 12 prospective studies conducted in the United Kingdom, Germany, Singapore, Japan, and Sweden, involving more than one million treatments. He estimated the risk of acupuncture-related adverse events at 0.05 per 10,000 treatments and 0.55 per 10,000 individual patients [19]. The findings from these studies indicate that the risks associated with acupuncture are minimal, a conclusion supported by Vincent [10], who stated that acupuncture is safe when performed by an experienced practitioner.

Minor side events related to cosmetic acupuncture, such as bleeding, ecchymosis, and infection, have been reported during the treatment phase. If there is bleeding after the needle is withdrawn, the area is pressed with cotton wool for one minute to prevent bruising. Should bruising or infection occur, Arnica cream or an oral antibiotic (e.g., cephalexin) will be used, respectively.

No special care is required during the treatment and follow-up periods, and participants may continue their daily activities, including swimming, without restriction.

12. Ethics

The trial protocol adheres to the principles outlined in the Declaration of Helsinki and was approved by the Mashhad University of Medical Sciences Ethics Committee on 7 April 2023 (Ethics Reference No: IR.MUMS.REC.1402.011).

Any modifications to the protocol that may affect the conduct of the study, potential participant benefits, or participant safety—including changes to study objectives, primary outcomes, sample size calculations, eligibility criteria, methods of allocation concealment, blinding, statistical analysis, interventions, or important administrative aspects—require a formal protocol amendment. These amendments must first be approved by the TSC and DMC before being submitted for registration with the IRCT. If necessary, the protocol amendment must also be approved by the Mashhad University of Medical Sciences Ethics Committee before implementation.

13. Dissemination plans

The results of this trial will be published as an original article in an ISI-indexed journal, which will be made available to the international scientific community.

The GLS and the QOL scores (SF-36 questionnaire) will be assessed in both groups at baseline (week 0), the post-treatment phase (week 7), and the follow-up phase (week 12). The SSS will be assessed in both groups at week 7 (post-treatment phase) and week 12 (follow-up phase). The GAIS will be calculated based on the difference in GLS scores at the post-treatment (week 7) and follow-up (week 12) phases, compared to the baseline (week 0) for each participant.

To the best of our knowledge, this is the first clinical trial to investigate the effects of body and facial acupuncture on frown lines. This study is designed as a double-arm, randomized waitlist-controlled trial, with outcome assessors and the data analyst blinded to the group assignments. The selection of facial and body acupoints is based on established protocols in cosmetic and facial rejuvenation acupuncture literature [8,20-22], with consensus from expert acupuncturists.

Moisturizing the skin is widely considered to be the first step in anti-aging skincare. Maintaining the balance of water content, fat, and natural moisturizing factors in the stratum corneum is crucial for maintaining skin hydration and elasticity [23]. As part of the aging process, repeated contractions of facial muscles lead to the appearance of dynamic wrinkles [24]. The mechanism of acupuncture’s effects can be categorized as local and systemic effects. Locally, acupuncture induces micro-injuries from intradermal needles placed in the frown line, which can promote skin hydration by improving local blood circulation [6,8,25].

Preliminary evidence from at least one study suggests that cosmetic acupuncture may increase the water and fat content of facial skin [11]. Additionally, these micro-injuries can stimulate collagen production, filling in the frown line [20]. Another localized effect of acupuncture involves targeting the contracted facial muscles that cause frown lines. Inserting needles into these muscles may help to relax them [1,25].

Systemically, acupuncture has been shown to influence the limbic system by regulating endorphins and other neurotransmitters such as serotonin and dopamine, which are responsible for controlling emotions [25]. By influencing these neurotransmitters, acupuncture can help reduce muscle contractions associated with frowning, thereby helping to improve frown lines.

Since secondary outcomes in this study are subjective and self-reported by the participants, a limitation of this study is that they can be affected by the placebo effect or individual bias. To mitigate this limitation, the primary outcome will rely on standard objective measurements—photographic images assessed by three independent evaluators, which should assist in ensuring the validity and reliability of our results.

The results of this trial will provide comprehensive and reliable evidence of the efficacy and safety of acupuncture in reducing frown lines.

This work was supported by Mashhad University of Medical Sciences (grant number 4011855). Mashhad University of Medical Sciences will support only the cost of consumables, including the needles, used in the research.

HH: Conceptualization, Project administration, Writing- Original draft preparation. MJY: Conceptualization, Resources. MKhR: Methodology. FB: Writing- Reviewing and Editing. HA: Conceptualization, Supervision, Funding acquisition. All authors read and approved the final version.

The authors declare no conflict of interest.