lIfestyle iNterventionS for PaIn ReliEf (INSPIRE)

The Numerical Rate Score (NRS) will be used to assess the changes in the level of pain in response to the intervention between baseline and follow-up.

Participants reported their pain on a scale ranging from 0 to 10, where 0 represents no pain and 10 the worst pain imaginable.

Measures how many times a participant can rise from a chair to a full standing position in 30 seconds.

Time in seconds to stand up, walk 3m, return, and sit. Average of 3 trials at baseline and follow-up (at the end of 6 weeks).

Measured with dynamometer; average of 3 trials on dominant hand in seated position with participant applying as much grip pressure as possible on the dynamometer. The maximum reading (kg) in taken for each repetition.

Pain sensitisation will be measured using quantitative sensory testing (QST) to assess any changes in pain sensitisation indices from at baseline to follow-up. QST is a non-invasive method to assess pain sensitivity using standardised stimuli like mechanical pressure or sharpness. We used the QST modality temporal summation (TS). TS assesses sensitivity to sharpness by applying a brief "pinprick" stimulus (256 mN Pinprick; MRC-Systems, Heidelberg, Germany) to the skin with higher ratings potentially suggesting increased spinal cord pain sensitivity. A single stimulus was applied to the rectus femoris (5cm above the mid-point of the patella of the most painful knee), followed by 10 repetitive stimuli at 1/s. Participants rated pain/sharpness intensity on a 0-10 Visual Analogue Scale after both the single and the average of the repeated stimuli. Each test was performed twice, with a 2-minute break between repetitions and the average was taken.

Pain sensitisation will be measured using quantitative sensory testing (QST) to assess any changes in pain sensitisation indices from at baseline to follow-up. QST is a non-invasive method to assess pain sensitivity using standardised stimuli like mechanical pressure or sharpness. We used the QST modality pressure pain detection threshold (PPT) at anatomical position: quadricep (2cm above superolateral edge of patella). PPT measures the lowest pressure a participant perceives as painful while pressure is applied using a handheld probe (Medoc-AlgoMed, Israel) at a rate of 50 kPa/s on the most painful knee, with lower PPT threshold suggestive of increased pain sensitivity.

Pain sensitisation will be measured using quantitative sensory testing (QST) to assess any changes in pain sensitisation indices from at baseline to follow-up. QST is a non-invasive method to assess pain sensitivity using standardised stimuli like mechanical pressure or sharpness. We used the QST modality pressure pain detection threshold (PPT) at anatomical position: quadricep (2cm above superomedial edge of patella). PPT measures the lowest pressure a participant perceives as painful while pressure is applied using a handheld probe (Medoc-AlgoMed, Israel) at a rate of 50 kPa/s on the most painful knee, with lower PPT threshold suggestive of increased pain sensitivity.

Pain sensitisation will be measured using quantitative sensory testing (QST) to assess any changes in pain sensitisation indices from abeline to follow-up. QST is a non-invasive method to assess pain sensitivity using standardised stimuli like mechanical pressure or sharpness. We used the QST modality pressure pain detection threshold (PPT) at the anatomical position: medial joint line (3cm medially from medial edge of patella). PPT measures the lowest pressure a participant perceives as painful while pressure is applied using a handheld probe (Medoc-AlgoMed, Israel) at a rate of 50 kPa/s on the most painful knee, with lower PPT threshold suggestive of increased pain sensitivity.

Pain sensitisation will be measured using quantitative sensory testing (QST) to assess any changes in pain sensitisation indices from at baseline to follow-up. QST is a non-invasive method to assess pain sensitivity using standardised stimuli like mechanical pressure or sharpness. We used the QST modality pressure pain detection threshold (PPT) at anatomical position: tibialis anterior (5 cm distal and 1 cm lateral to the tibial tuberosity). PPT measures the lowest pressure a participant perceives as painful while pressure is applied using a handheld probe (Medoc-AlgoMed, Israel) at a rate of 50 kPa/s on the most painful knee, with lower PPT threshold suggestive of increased pain sensitivity.

Pain sensitisation will be measured using quantitative sensory testing (QST) to assess any changes in pain sensitisation indices from baseline to follow-up. QST is a non-invasive method to assess pain sensitivity using standardised stimuli like mechanical pressure or sharpness. We used the QST modality pressure pain detection threshold (PPT) at anatomical position: brachioradialis (5 cm medial and distal to the lateral epicondyle) on the arm opposite the painful knee. PPT measures the lowest pressure a participant perceives as painful while pressure is applied using a handheld probe (Medoc-AlgoMed, Israel) at a rate of 50 kPa/s on the arm opposite the painful knee, with lower PPT threshold suggestive of increased pain sensitivity.

Change in serum levels of short-chain fatty acid (SCFA) butyric acid in response to the interventions. SCFA levels in serum will be measured using mass spectrometry.

Change in serum levels of short-chain fatty acid (SCFA) acetic acid in response to theinterventions. SCFA levels in serum will be measured using mass spectrometry.

Change in Interleukin-6 (IL-6) inflammatory protein levels measured with the Olink for a subset of individuals. For Olink cytokine assay panels data is reported in either Normalized Protein eXpression units (NPX) units or absolute concentration units (pg/mL), with the lower limit of detection typically below 1 pg/mL for most assays.

Change in Tumor Necrosis Factor (TNF) inflammatory protein levels measured with the Olink for a subset of individuals. For Olink cytokine assay panels data is reported in either Normalized Protein eXpression units (NPX) units or absolute concentration units (pg/mL), with the lower limit of detection typically below 1 pg/mL for most assays.

Change in Interferon gamma (IFN-γ) inflammatory protein levels measured with the Olink for a subset of individuals. For Olink cytokine assay panels data is reported in either Normalized Protein eXpression units (NPX) units or absolute concentration units (pg/mL), with the lower limit of detection typically below 1 pg/mL for most assays.

The gut microbiome will be measured with shotgun metagenomic sequencing of stool samples and the Shannon Diversity Index will be calculated. The Shannon Diversity Index assesses both the richness (number of different species) and evenness (distribution of individuals among species) within the gut microbiome. A higher Shannon index indicates a more diverse microbiome, while a lower index suggests less diversity.

Serum levels of Endocannabinoid Anandamide (AEA) will be measured using mass spectrometry in samples collected at baseline and at follow-up (at the end of six weeks). This will help us ascertain the change in levels of Endocannabinoid Anandamide (AEA) in response to the intervention.

Serum levels of 2-arachidonoylglycerol (2-AG) Endocannabinoid will be measured using mass spectrometry in samples collected at baseline and at follow-up (at the end of six weeks). This will help us ascertain the change in levels of 2-arachidonoylglycerol (2-AG) Endocannabinoid in response to the intervention.

Transcriptomics will be measured using RNA sequencing of blood samples collected at baseline and at follow-up (at the end of six weeks). RNA was extracted from whole blood, and sequencing libraries were prepared and analysed using standard RNA-seq pipelines.

Transcriptomics will be measured using RNA sequencing of blood samples collected at baseline and at follow-up (at the end of six weeks). RNA was extracted from whole blood, and sequencing libraries were prepared and analysed using standard RNA-seq pipelines.

Transcriptomics will be measured using RNA sequencing of blood samples collected atbaseline and at follow-up (at the end of six weeks). RNA was extracted from whole blood,and sequencing libraries were prepared and analysed using standard RNA-seq pipelines.