The efficacy of alpha-lactalbumin supplementation for improving sleep quality, mood and cognition in active individuals with sleep difficulties

Study design -The study will implement a mixed factorial, placebo-controlled, crossover RCT to test the efficacy of evening alpha-lactalbumin (ALAC) supplementation across varying dietary protein intake (low protein (LPD) v/s habitual protein diet (HPD)) for improving sleep quality and morning-after mood, alertness and cognition in healthy active individuals with existing sleep difficulties. Therefore the arms of the study include: 1) Arm 1: HPD-ALAC 2) Arm 2: HPD-Placebo 3) Arm 3: LPD-ALAC and 4) Arm 4: LPD-Placebo Randomisation: Participants will be randomly allocated to a diet group, either a habitual protein diet (HPD) OR low protein diet (LPD) in a counterbalanced manner. Both participants and researchers will not be blinded to the diet group allotment but will be blinded to the experimental drinks received (ALAC and Placebo). For blinding purposes, both the experimental drinks will be matched for quantity, taste, and appearance and be further coded as A & B. The treatment order to the participants will be randomly allocated via a randomisation list generated using Stata/SPSS. The uncoded data regarding the allocated treatment order and the drinks for (placebo or ALAC) will be stored securely with limited access to only the principal investigator of this research team until after data analysis is complete. Baseline: Upon screening, eligible participants will undergo a baseline sleep, mood and cognition testing. Sleep testing will be conducted for 3 nights in the participant's home, During baseline testing of 3 nights, everyday participants will be asked to: -Record their 24-hr diet recall via smartphone app (Easy Diet Diary – Xyris Pty Ltd, Brisbane, QLD), -Complete the KSS questionnaire (Appendix D) 60-minutes and 30-minutes before habitual bedtime. - Wear the wrist actigraphy (Actical Z series) watch on non-dominant hand, - Place the LUX monitor close to eye level on clothing/ pillow cover, - Use the Somfit forehead patch with electrodes (as per instruction booklet provided) - Record the time-to-bed in the online sleep diary (via a QR code provided to them in instruction booklet). - Upon waking up each morning, complete the online sleep diary - Complete the KSS questionnaire within 1 hour of waking up. After completing three nights of baseline, next morning: - Collect first morning urine sample, store in refrigerator/ cool dark place and carry to Deakin lab, - Complete BRUMS, Morningness -Eveningness (Chronotype), Sleep Hygiene Index (SHI) questionnaire - Visit the Deakin University, Burwood laboratory, within 3 hours of waking, for an alertness and cognition testing. Intervention: This phase will also occur primarily in participant's home environment. Diet Standardisation for diet groups: Participants will be allocated to either a habitual protein diet (HPD) group or low protein diet (LPD) group in a random order. The total daily energy intake will be determined individually for each participant based on their average habitual dietary intake recorded at baseline. Food allergies (if any) will be clearly captured in the online screening questionnaire and at baseline, to ensure meals catered or meal guidelines to participants do not include specified allergens. Meals and guidelines will majorly try to include foods commonly featuring in the participant's habitual dietary intake at baseline. Any dietary preferences will be confirmed with participants at their baseline testing session at Deakin lab. -LPD diet will comprise of a protein intake of 0.8-1g/kg/d and a macronutrient (Carbohydrate: Protein: Fat ratio) percentage distribution of approximately (55:10:35) and a restricted protein intake at dinner (approximately 0.1-0.2g/kg/d) -HPD diet will comprise a protein intake of 1.4-2.0g/kg/d and macronutrient (Carbohydrate: Protein: Fat ratio) percentage distribution of (55:25:20) and an even distribution of protein across all meals. All participants will be provided with breakfast and dinner meals and a protein drink for the total of six nights across the intervention (3 nights per treatment condition). The meals provided will be as per their assigned diet group. Additionally, meal plan guidelines will be provided to follow strictly for lunch and snack meals (not be provided by the researchers). Food items outside of the guidelines will not be allowed to be consumed. Participants will require to log their lunch and snacks consumed in the EDD (Easy Diet Diary – Xyris Pty Ltd, Brisbane QLD) mobile app to confirm adherence to the diet protocol. Both, the participants and researchers will be aware to the diet group allocation (non-blinded). Participants will be additionally be provided with a protein drink (double-blinded) to be consumed once daily, for three nights of each treatment condition (ALAC or Placebo) during the intervention. Participants will be instructed to consume the protein drinks 180-minutes before their habitual bedtime. During 3 nights of each treatment condition (ALAC or Placebo), everyday participants will be asked to: -Record their 24-hr diet recall via smartphone app (Easy Diet Diary – Xyris Pty Ltd, Brisbane, QLD), -Complete the KSS questionnaire (Appendix D) 60-minutes and 30-minutes before habitual bedtime. - Wear the wrist actigraphy (Actical Z series) watch on non-dominant hand, - Place the LUX monitor close to eye level on clothing/ pillow cover, - Use the Somfit forehead patch with electrodes (as per instruction booklet provided) - Record the time-to-bed in the online sleep diary (via a QR code provided to them in instruction booklet). - Upon waking up each morning, complete the online sleep diary - Complete the KSS questionnaire within 1 hour of waking up. After completing three nights of baseline, next morning: - Collect first morning urine sample, store in refrigerator/ cool dark place and carry to Deakin lab, - Complete BRUMS - Visit the Deakin University, Burwood laboratory, within 3 hours of waking, for an alertness and cognition testing. In a crossover manner, participants will complete both the treatment conditions (ALAC or Placebo) of three nights each. The order of receiving treatments will be randomised and each treatment condition (ALAC or Placebo) will be separated by a washout period of minimum of five-nights to limit carryover effect. Protein drink: The protein drinks (treatments) will either contain 40g of experimental ALAC protein (BiPRO Alpha 9000; Agropur, US) or 40g of placebo protein (Glycomacropeptide BiPRO GMP; Agropur, US), mixed with 25g of sugar-free drinking chocolate powder reconstituted in 250ml water. Both the drinks will be identical in appearance and matched for taste to ensure double blinding of the treatment. The drinks will be coded by an external researcher who is not involved actively in data collection and analysis, ensuring double-blinding of the treatment conditions. Strategies used to assess adherence: Sleep will be monitored using Somfit dry-EEG devices and wrist actigraphy, ambient light will be monitored overnight using LYS light buttons, adherence to diet allocation and protein consumption will be monitored using Easy diet diary mobile app where participants will log their meals consumed with images of the same. Participants will also complete sleep and training logs daily during the intervention and answer questionnaires on mood rating and alertness. Post each treatment condition (3 nights), participants will visit Deakin university lab in the morning (similar to baseline) for cognition testing. Each session at Deakin Uni will last approx. 1 hour. As is the nature of a crossover trial, each participant will serve as their own control.