The Discovery Academy, in Bentilee, Stoke-on-Trent, made the controversial changes this week.
Parents of pupils claim the barriers are locked during lesson times.
The issue of periods, bowel conditions and the potential impact of unpredictable upset stomachs have all been raised.
On Thursday, more than 70 pupils walked out of classes in protest of the new decision.
One mum, 30, said: ‘I was flicking through Snapchat when I saw one of the gates. I asked my son “are there gates on the toilets?” He said “yes”.
‘I phoned the office and asked them about it. I said “send my son home now”. They asked me why and I told them “because I said so, that’s why”.
‘My son asked to go to the toilet and he was told “no”. He wasn’t allowed and he has a medical pass.
‘It’s hard enough being a girl at that age never mind bleeding through in front of your whole class. I’m not having my daughter humiliated.
‘With periods young girls don’t know when they are coming. You try to prepare as much as possible but there are still incidents.
‘You can’t prepare for an upset stomach, I think it’s disgusting.’
The Alpha Academies Trust, which runs The Discovery Academy, has not commented on who is able to get a medical pass for the toilets.
The Discovery Academy, in Bentilee
Children are reportedly able to get medical passes which allow them to use the bathrooms whenever they like.
It is not clear who qualifies for a medical pass.
A spokesperson said: ‘At AAT we have high expectations of learning and clear processes in place to support.
‘We have high standards of uniform ready for the world ahead and have processes in place for those who truant.
‘We encourage students to go to the toilet at regular set times during the day and support those with medical needs by accessing toilets at any time.
‘Parental engagement is extremely important to us and we value opportunities to listen to concerns and explain the reasons for our decisions.’