## 5 Materials and Methods
### Registration and Protocol  
The protocol of this systematic review was registered in the international prospective register of systematic reviews (PROSPERO) database (registration number: CRD42023432411) [48]. Deviations from the published protocol are as follows: (1) Inclusion of \"systematic review\" in the title to follow PRISMA 2020 guidelines. (2) We added to the search strategy other sources such as trials and systematic reviews registries. (3) Given the poor definition of chronicity in the available disease models, the eligibility criteria was updated, and we excluded studies when it was unclear if they were related to acute or chronic conditions. (4) We have excluded studies evaluating combined therapies, except when data were available for BoNT key effects only. (5) We have excluded disease models (or assessments) reported below head and neck. (6) To assess the quality of evidence for each pre-specified outcome, we considered the GRADE approach. (7) Given the number of studies presenting measurements only in graphs, we have attempted to extract data with ImageJ software. (8) We have obtained the collaboration of an additional author. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/toxins17080377/s1](https://www.mdpi.com/article/10.3390/toxins17080377/s1)]([https://www.mdpi.com/article/10.3390/toxins17080377/s1](https://www.mdpi.com/article/10.3390/toxins17080377/s1)), File S1: Raysan report; File S2, Table S1: Biomarkers in Preclinical Research on Botulinum Toxin effects on Chronic Inflammatory State. Listed reasons for contacting the corresponding author as per protocol [35; 36; 37; 38; 39; 40]; File S3, Table S2: Summary of findings [13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34]; File S4, Table S3: Risk of bias assessment--SYRCLE's tool [13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 27; 29; 31; 32; 34]; File S5, Table S4: Summary of findings for the biomarkers reported in only one of the included studies [13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 25; 27; 29; 31; 32; 34]; File S6, Table S5: GRADE assessment for biomarkers reported in single experiments [13; 14; 15; 16; 17; 18; 20; 21; 22; 23; 25; 27; 29; 31; 32; 33]; File S7, Table S6: Synopsis of outcomes [13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 25; 27; 29; 31; 32; 34]; File S8: GRADE consistency assessment [13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34]; File S9 PRISMA 2020 checklist; File S10: Table S7 reviewer 1 (I.N.P.) search strategy; File S10 Table S8 reviewer 2 (S.D.) search strategy. **Author Contributions:** I.N.P., G.D.I.T.C., S.D., R.S., A.C.B., A.M.A., H.H., A.C.M. and R.F.-A. were involved in the study conception and design. Material preparation, data extraction, and analysis were performed by I.N.P., S.D., G.D.C., R.S., H.H. and I.N.P. wrote the first draft of the manuscript, and I.N.P., G.D.C., S.D., R.S., A.C.B., A.M.A., H.H., A.C.M. and R.F.-A. commented and provided critical revisions. All authors have read and agreed to the published version of the manuscript. **Funding:** This work was supported by a grant from FCT--Fundacao para a Ciencia e a Tecnologia I.P. (2023.00543.BD) and DOI [[https://doi.org/10.54499/2023.00543.BD](https://doi.org/10.54499/2023.00543.BD)]([https://doi.org/10.54499/2023.00543.BD](https://doi.org/10.54499/2023.00543.BD)) as a Doctoral Scholarship to I.N.P. **Institutional Review Board Statement:** Not applicable. **Informed Consent Statement:** Not applicable. **Data Availability Statement:** The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author. For example, the full list of citations that were excluded and recorded in the Rayyan platform or the listed reasons for inclusion versus exclusion studies requiring adjudication regarding eligibility are available upon request.