The objective of the present study is to assess the rates of acquired tolerance to cow's milk (CM) after 36 months in subjects who consumed amino acid‐based formula with synbiotics (AAF‐S) or amino acid‐based formula without synbiotics (AAF) during a 1‐year intervention period in early life as part of the PRESTO study (Netherlands Trial Register number NTR3725). Differences in CM tolerance development between groups were analysed using a logistic regression model. Results show that the proportion of subjects (mean ±SD age, 3.8 ± 0.27 years) who developed CM tolerance after 36 months was similar in the group receiving AAF‐S (47/60 78%) and in the group receiving AAF (49/66 74%) (p = 0.253), that is, figures comparable to natural outgrowth of CM allergy. Our data suggest that the consumption of AAF and absence of exposure to CM peptides do not slow down CM tolerance acquisition. What is Known The randomized double‐blind controlled trial PRESTO showed that infants receiving amino acid‐based formula (AAF) with or without synbiotics for 12 months in early life demonstrated effective symptom resolution. After 12 and 24 months, development of tolerance to cow's milk (CM) was in line with natural outgrowth. What is New This study shows that, 36 months after the start of the intervention, a high proportion of children (about three‐quarters) developed tolerance to CM. These data suggest that the consumption of AAF, with or without synbiotics, and the absence of exposure to CM peptides do not slow down CM tolerance acquisition.