Aims/hypothesis <p>This study aimed to investigate how pre-meal whey protein (WP) supplementation throughout the third trimester of pregnancy affects glycaemic and metabolic outcomes in women with gestational diabetes mellitus (GDM). The hypothesis was that WP, when administered as a pre-meal 30 min before breakfast daily, lowers glycaemic variability (primary outcome: CV%).</p> Methods <p>In a double-blinded, randomised, placebo-controlled, parallel trial, 62 women with GDM were randomised to receive 20 g WP isolate/day or placebo 30 min before breakfast throughout the third trimester. Participants were randomly assigned (<a href="http://www.randomiser.org">www.randomiser.org</a>) to WP or placebo using a computer-generated list. Allocation was concealed with sealed strips. Participants, caregivers, investigators and outcome assessors were masked, except the dietitian providing dietary guidance. Eligibility criteria included GDM, normotension and age ≥18 years. Exclusion criteria included special dietary regimens ≥1 month, daily protein supplements, food allergies, glucose-metabolism-affecting drugs, twin pregnancies, polycystic ovary syndrome, severe comorbidity, hyperemesis or non-breakfast eaters. The study included laboratory visits, home-based measurements under controlled-living and free-living conditions during the early and late third trimester, and follow-up at delivery. Glucose levels were assessed using continuous glucose monitoring.</p> Results <p>A total of 29 women were randomised to placebo and 33 were randomised to WP, with 25 in the placebo group and 30 women in the WP group completing the study. In the WP group, the 1 h postprandial glucose following breakfast was −20% (95% CI −28%, −11%) lower in the early and −15% (95% CI −24%, −5%) lower in the late third trimester compared with the placebo group under controlled conditions. Similarly, the 1 h postprandial glucose was −14% (95% CI −23%, −4%) lower in the early and −8% (95% CI −18%, 3%) lower in the late third trimester under free-living conditions. Glycaemic variability was lower in the WP group under controlled-living conditions. The mean amplitude of glycaemic excursions (MAGE) was lower during both the early and late third trimester, and the SD and CV% were lower during the early third trimester (all <i>p</i>&lt;0.05). Time in range (proportion of time spent with glucose levels 3.5–7.8 mmol/l) was lower during free-living in the late third trimester (<i>p</i>=0.05).</p> Conclusions/interpretation <p>Pre-meal WP improves glycaemic management and reduces glucose variability in women with GDM under controlled-living and free-living conditions. Future research should evaluate whether WP can delay or prevent pharmacological treatments such as insulin initiation.</p> Trial registration <p>ClinicalTrials.gov NCT04767880</p> Funding <p>Department of Clinical Medicine, Aarhus University and Arla Foods Ingredients Group P/S (Agr-2020–731–12107).</p> Graphical Abstract <p></p>