People with obesity often struggle to maintain their weight loss after a weight loss period. Furthermore, the effect of weight loss on appetite and food preferences remains unclear. Hence this study investigated the effect of weight loss on subjective appetite and food preferences in healthy, overweight and obese volunteers. A subgroup of adult participants (n = 123) from the Diet Obesity and Genes (DiOGenes) study (subgroup A) was recruited from across six European countries. Participants lost ≥8% of initial body weight during an 8-week low calorie diet (LCD). Subjective appetite and food preferences were measured before and after the LCD, in response to a standardized meal test, using visual analogue rating scales (VAS) and the Leeds Food Choice Questionnaire (FCQ). After the LCD, participants reported increased fullness (p < 0.05), decreased desire to eat (p < 0.05) and decreased prospective consumption (p < 0.05) after consuming the test meal. An interaction effect (visit x time) was found for hunger ratings (p < 0.05). Area under the curve (AUC) for hunger, desire to eat and prospective consumption was decreased by 18.1%, 20.2% and 21.1% respectively whereas AUC for fullness increased by 13.9%. Preference for low-energy products measured by the Food Preference Checklist (FPC) decreased by 1.9% before the test meal and by 13.5% after the test meal (p < 0.05). High-carbohydrate and high-fat preference decreased by 11.4% and 16.2% before the test meal and by 17.4% and 22.7% after the meal (p < 0.05). No other effects were observed. These results suggest that LCD induced weight loss decreases the appetite perceptions of overweight volunteers whilst decreasing their preference for high-fat-, high-carbohydrate-, and low-energy products.